Transaminase reactions

ABSTRACT

The present disclosure relates to methods of using transaminase polypeptides in the synthesis of chiral amines from prochiral ketones.

This application is a Continuation of co-pending U.S. patent applicationSer. No. 14/547,339, filed Nov. 19, 2014, which is a Divisionalapplication of U.S. patent application Ser. No. 13/378,963, filed Apr.9, 2012, now U.S. Pat. No. 8,921,079, which is a 371 filing of PCTInternational Application No. PCT/US2010/039343, filed Jun. 21, 2010,which claims benefit of U.S. Provisional Application No. 61/219,372,filed Jun. 22, 2009, and U.S. Provisional Application No. 61/308,873,filed Feb. 26, 2010, the contents of each of which are incorporatedherein by reference.

TECHNICAL FIELD

The present disclosure relates to transaminase biocatalysts and methodsof using the biocatalysts.

REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM

The official copy of the Sequence Listing is submitted concurrently withthe specification as a 359 kbyte ASCII formatted file,“CX2-019WO1_ST25.txt,” created Jun. 21, 2010. The Sequence Listing isfiled via EFS-Web as part of the specification and is herebyincorporated in its entirety by reference herein. The Sequence Listingfile is identical except for minor formatting changes to the 367 kbyteASCII formatted Sequence Listing file “376247-042USP1.txt” created onFeb. 26, 2010 which was incorporated by reference in the priority U.S.provisional application 61/308,873.

BACKGROUND

Aminotransferases, also known as transaminases (E.C. 2.6.1) catalyze thetransfer of an amino group, a pair of electrons, and a proton from aprimary amine of an amino donor substrate to the carbonyl group of anamino acceptor molecule.

A general transaminase reaction is shown in Reaction I, below. In thisreaction, an amino acceptor (keto, or ketone) which is the precursor ofthe desired amino acid product, is reacted with an amino donor. Thetransaminase enzyme exchanges the amino group of the amino donor withthe keto group of the amino acceptor. The reaction therefore results inthe desired chiral amine product and a new amino acceptor (keto)compound, which is a by-product.

An exemplary stereoselective transamination by a transaminase isdemonstrated by the activity of transaminase from Arthrobacter sp.KNK168 on 3,4-dimethoxyphenylacetone (see e.g., Iwasaki et al., 2006,Appl. Microbiol. Biotechnol. 69: 499-505; and U.S. Pat. No. 7,169,592,each of which is hereby incorporated by reference herein). Thus,transaminase enzymes have potential industrial use for stereoselectivesynthesis of optically pure chiral amines and the enantiomericenrichment of chiral amines and amino acids (Shin et al., 2001, Biosci.Biotechnol. Biochem. 65:1782-1788; Iwasaki et al., 2003, Biotech. Lett.25:1843-1846; Iwasaki et al., 2004, Appl. Microb. Biotech. 69:499-505,Yun et al., 2004, Appl. Environ. Microbiol. 70:2529-2534; and Hwang etal., 2004, Enzyme Microbiol. Technol. 34: 429-426). Chiral amines playan important role in the pharmaceutical, agrochemical and chemicalindustries. Chiral amines are frequently used as intermediates orsynthons for the preparation of various pharmaceuticals, such ascephalosporin or pyrrolidine derivatives. Examples of the use ofaminotransferases to generate useful chemical compounds include:preparation of intermediates and precursors of pregabalin (e.g., WO2008/127646); the stereospecific synthesis and enantiomeric enrichmentof β-amino acids (e.g., WO 2005/005633); the enantiomeric enrichment ofamines (e.g., U.S. Pat. No. 4,950,606, U.S. Pat. No. 5,300,437, and U.S.Pat. No. 5,169,780); and the production of amino acids and derivatives(e.g., U.S. Pat. No. 5,316,943, U.S. Pat. No. 4,518,692, U.S. Pat. No.4,826,766, U.S. Pat. No. 6,197,558, and U.S. Pat. No. 4,600,692). Hence,transaminases are useful for the enantiomeric enrichment andstereoselective synthesis of chiral amines

SUMMARY

The present disclosure provides processes for the biocatalyticconversion of prochiral keto substrates to chiral amines in presence ofan amino group donor, and transaminase biocatalysts and correspondingpolynucleotides for use in the processes. In one aspect, the presentinvention comprise a process for preparing an amine product ofstructural formula (I):

having the indicated stereochemical configuration at the stereogeniccenter marked with an *; in enantiomeric excess over the oppositeenantiomer, wherein

R¹ optionally substituted aryl or heteroaryl; and

R² is an optionally substituted C₁-C₆ alkyl, —R³C(O)R⁴, or —R³OC(O)R⁵;

wherein R³ is an optionally substituted C₁-C₄ alkyl; and R⁴ is H, anoptionally substituted C₁-C₄ alkyl, NR⁶R⁷, or OR⁸, where R⁵, R⁶, R⁷, andR⁸ are independently H or C₁-C₄ alkyl, which process comprisescontacting a ketone substrate of structural formula (II):

with a transaminase polypeptide in presence of an amino donor underreaction conditions suitable for converting the ketone substrate to theamine product. In some embodiments, the transaminase polypeptide has atleast 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more amino acid sequence identity to SEQ ID NO:4 and iscapable of converting the ketone substrate to the amine product at arate that is improved as compared to the transaminase of SEQ ID NO:2.

In some embodiments, the transaminase polypeptide has at least 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore amino acid sequence identity to SEQ ID NO: 58, 72, 74, 80, 86, 96,98, 100, or 102 and is capable of converting the ketone substrate to theamine product at a rate that is improved as compared to the transaminaseof SEQ ID NO:2. Specific embodiments of the engineered transaminases foruse in the processes are further provided in the detailed description.

In some embodiments, the engineered transaminase has at least 5%, 10%,20%, 30%, 40%, 50% or more activity of SEQ ID NO:74.

In any of the embodiments of the processes disclosed herein, the processcan be carried out wherein the transaminase polypeptide is capable ofconverting the sitagliptin ketoamide substrate to sitagliptin at a ratethat is improved as compared to the transaminase of SEQ ID NO:2, and hasat least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more amino acid sequence identity to SEQ ID NO:4.

In some embodiments, the amine product is produced in at least 70%, 80%,85%, 90%, 95%, 96%, 97%, 96%, or 99% or more enantiomeric excess. Insome embodiments of the processes described above, the amine product isproduced in at least 99% enantiomeric excess.

In some embodiments of the process, R¹ is an optionally substitutedphenyl. In some embodiments, R¹ is an optionally substituted pyridinyl.In some embodiments, the substitutions on the aryl or heteroaryl areselected from C₁-C₄ alkyl, —OR′, —SR′, —NR′R′, —NO₂, —NO, —CN, —CF₃,halogen (e.g., —F, —Cl, —Br and —I), —C(O)R′, —C(O)OR′, —C(O)NR′,—S(O)₂R′, —S(O)₂NR′R″, where each R′ and R″ are independently selectedfrom the group consisting of hydrogen and (C₁-C₄) alkyl. In someembodiments R² is methyl or halo substituted methyl. In someembodiments, R² is CF₂H or CF₃.

In some embodiments, the substitution on C₁-C₆ alkyl and R³ of group R²are selected from halogen, NR⁵R⁶ or —OR⁸, where R⁵ and R⁶ are definedabove and R⁸ is H or C₁-C₄ alkyl. In some embodiments, the compounds offormula (I) produced in the process described above are substantiallychirally pure compounds. In certain embodiments, the compounds offormula (I) produced in the process described above are chirally pure.

In some embodiments of the process, the amine product of formula (I) is:

wherein R⁹ is H, Cl, Br, F, CH₃, CF₃, NH₂, NO₂, CN, SCN, or OCH₃, and R²is an optionally substituted C₁-C₆ alkyl, and the ketone substrate offormula (II) is:

In some embodiments of the process, the amine product of formula (I) is:

wherein R⁹ is H, Cl, Br, F, CH₃, CF₃, NH₂, NO₂, CN, SCN, or OCH₃, andthe ketone substrate of formula (II) is:

In some embodiments R⁹ is Br, CH₃, or CF₃. In some embodiments, R⁹ is inthe para position on the phenyl ring.

In some embodiments of the process, the amine product(S)-1-(4-bromophenyl)-2,2,2-trifluoroethanamin is prepared inenantiomeric excess from 1-(4-bromophenyl)-2,2,2-trifluoroethanone:

In some embodiments of the process, the amine product(S)-2,2,2-trifluoro-1-p-tolylethanamine is prepared in enantiomericexcess from 2,2,2-trifluoro-1-p-tolylethanone:

In some embodiments of the process, the amine product(S)-2,2,2-trifluoro-1-(4-(trifluoromethyl)phenyl)ethanamine is preparedin enantiomeric excess from2,2,2-trifluoro-1-(4-(trifluoromethyl)phenyl)ethanone:

In some embodiments of the process, the amine product of formula (I) is:

and the ketone substrate of formula (II) is:

wherein R⁷ is optionally substituted C₁-C₄ alkyl, and R¹⁰ is R⁹ definedabove. In some embodiments, R¹⁰ is H or F. In some embodiments, R⁷ isC₁-C₄ alkyl.

In some embodiments of the process, the amine product(R)-ethyl-3-amino-3-(pyridin-2-yl)propanoate is prepared in enantiomericexcess from ethyl 3-oxo-3-(pyrindin-2-yl)propanoate:

In some embodiments of the process, the amine product of formula (I) is:

wherein R¹¹ is halogen, OH, —C(O)R⁴, —OC(O)R⁵, or NR⁶R⁷, wherein R⁴, R⁵,R⁶, R⁷, and R¹⁰ are defined above, and the ketone substrate of formula(II) is:

In some embodiments of the process, the amine product(S)-4-chloro-1-(2-fluorophenyl)butan-1-amine is prepared in enantiomericexcess from 4-chloro-1-(2-fluorophenyl)butan-1-one:

In some embodiments, the disclosure provides a process for preparing thecompound of formula (III):

having the indicated stereochemical configuration at the stereogeniccenter marked with an *, in enantiomeric excess over the oppositeenantiomer, wherein R¹⁰ is as defined above, which process comprises:

(a) contacting a ketone substrate of formula:

wherein R¹¹ and R¹⁰ are as defined above, with a transaminasepolypeptide described herein in presence of an amino donor underreaction conditions suitable for converting the ketone substrate to anamine product of formula:

and

(b) cyclizing the amine product under suitable conditions to form thecompound of formula (III).

In some embodiments, the disclosure provides a process for preparing(R)-2-(2-fluorophenyl)pyrrolidine:

which process comprises converting ketone substrate4-chloro-1-(2-fluorophenyl)butan-1-one to the amine product(S)-4-chloro-1-(2-fluorophenyl)butan-1-amine in enantiomeric excess:

and cyclizing the (S)-4-chloro-1-(2-fluorophenyl)butan-1-amine to form(R)-2-(2-fluorophenyl)pyrrolidine:

in enantiomeric excess.

Various engineered transaminases can be used for carrying out theprocesses are described in the detailed description. In someembodiments, the process can use the engineered transaminasesrepresented by SEQ ID NO: 58, 72, 74, 80, 86, 96, 98, 100, or 102.

Any suitable amino donor can be used for the transamination reaction. Insome embodiments, the amino group donor is selected from isopropylamine,alanine, 3-aminobutyric acid, or methylbenzylamine. A preferred aminogroup donor is isopropylamine.

As noted herein, the processes described herein are carried out inreaction conditions suitable for converting the ketone substrate to thecorresponding chiral amine product in enantiomeric excess. In someembodiments, the reaction condition comprises a temperature of 20° C. to65° C. In some embodiments, the reaction condition comprises atemperature of 40° C. to 65° C. In some embodiments, the reactioncondition comprises a temperature of 50° C. to 65° C.

In some embodiments, the reaction condition under which the processesare carried out comprises a pH of about 7.0 to about 11.0. In someembodiments, the reaction condition comprises a pH of about 7.0 to about9.0. In some embodiments, the reaction condition for the process is a pHof about 8.5.

Various organic solvents can be used in the process to facilitate theenzyme reaction and to place the substrate and/or product in solution.In some embodiments of the process, the organic solvent comprises apolar solvent, such as methanol or dimethylsulfoxide (DMSO). In someembodiments, the organic solvent is DMSO, which can be present fromabout 10% to about 40% volume/volume (v/v). In some embodiments, theDMSO is present at about 40% v/v.

DETAILED DESCRIPTION

The present disclosure provides processes for converting certainprochiral keto substrates to chiral amine products using stereoselectiveengineered transaminase polypeptide biocatalysts. Embodiments of variousprocesses and the transaminases are described herein.

As used in this specification and the appended claims, the singularforms “a”, “an” and “the” include plural referents unless the contextclearly indicates otherwise. Thus, for example, reference to “a protein”includes more than one protein, and reference to “a compound” refers tomore than one compound.

Moreover, the use of “or” means “and/or” unless stated otherwise.Similarly, “comprise,” “comprises,” “comprising” “include,” “includes,”and “including” are interchangeable and not intended to be limiting.

It is to be further understood that where descriptions of variousembodiments use the term “comprising,” those skilled in the art wouldunderstand that in some specific instances, an embodiment can bealternatively described using language “consisting essentially of” or“consisting of”.

Abbreviations and Definitions

For the purposes of the descriptions herein, the abbreviations used forthe genetically encoded amino acids are conventional and are as follows:

Amino Acid Three-Letter Abbreviation One-Letter Abbreviation Alanine AlaA Arginine Arg R Asparagine Asn N Aspartate Asp D Cysteine Cys CGlutamate Glu E Glutamine Gln Q Glycine Gly G Histidine His H IsoleucineIle I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe FProline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine TyrY Valine Val V

When the three-letter abbreviations are used, unless specificallypreceded by an “L” or a “D” or clear from the context in which theabbreviation is used, the amino acid may be in either the L- orD-configuration about α-carbon (Cα). For example, whereas “Ala”designates alanine without specifying the configuration about the αcarbon, “D-Ala” and “L-Ala” designate D-alanine and L-alanine,respectively. When the one-letter abbreviations are used, upper caseletters designate amino acids in the L-configuration about the α-carbonand lower case letters designate amino acids in the D-configurationabout the α-carbon. For example, “A” designates L-alanine and “a”designates D-alanine. When peptide sequences are presented as a stringof one-letter or three-letter abbreviations (or mixtures thereof), thesequences are presented in the N→C direction in accordance with commonconvention.

Definitions

The technical and scientific terms used in the descriptions herein willhave the meanings commonly understood by one of ordinary skill in theart, unless specifically defined otherwise. Accordingly, the followingterms are intended to have the following meanings.

“Aminotransferase” and “transaminase” are used interchangeably herein torefer to a polypeptide having an enzymatic capability of transferring anamino group (NH₂) and a hydrogen atom from a primary amine (2) to anacceptor carbonyl (keto) compound (1), converting the amine donor intoits corresponding carbonyl (keto) compound (4) and converting theacceptor into its corresponding primary amine (3).

“Protein”, “polypeptide,” and “peptide” are used interchangeably hereinto denote a polymer of at least two amino acids covalently linked by anamide bond, regardless of length or post-translational modification(e.g., glycosylation, phosphorylation, lipidation, myristilation,ubiquitination, etc.). Included within this definition are D- andL-amino acids, and mixtures of D- and L-amino acids.

“Amino group donor” refers to an amino compound which is capable ofdonating an amino group to an acceptor carbonyl compound (i.e., an aminogroup acceptor), thereby becoming a carbonyl by-product. Amino groupdonors are molecules of general formula:

in which each of R^(C), R^(D), when taken independently, is an alkyl, analkylaryl group, or aryl group which is unsubstituted or substitutedwith one or more enzymatically acceptable groups. R^(C) can be the sameor different from R^(D) in structure or chirality. The groups R^(C) andR^(D), taken together, may form a ring that is unsubstituted,substituted, or fused to other rings. Typical amino group donors thatcan be used with the invention include chiral and achiral amino acids,and chiral and achiral amines

“Chiral amine” refers to amines of general formula R^(X)—CH(NH₂)—R^(Y)wherein R^(X) and R^(Y) are nonidentical and is employed herein in itsbroadest sense, including a wide variety of aliphatic and alicycliccompounds of different, and mixed, functional types, characterized bythe presence of a primary amino group bound to a secondary carbon atomwhich, in addition to a hydrogen atom, carries either (i) a divalentgroup forming a chiral cyclic structure, or (ii) two substituents (otherthan hydrogen) differing from each other in structure or chirality.

“Substrate” as used herein refers to an amino group acceptor, such as aketone, that accepts the amino group from an amino group donor in areaction mediated by a transaminase. In the context of the presentdisclosure, substrate for the transaminase includes, among others, thecompound of formula (II), as further described herein.

“Amino acceptor” and “amine acceptor”, “keto substrate”, “keto” and“ketone” are used interchangeably herein to refer to a carbonyl (keto,or ketone) compound which accepts an amino group from a donor amineAmino acceptors are molecules of general formula,

in which each of R^(A) and R^(B) in some embodiments, when takenindependently, can be an alkyl, an alkylaryl group, or aryl group whichis unsubstituted or substituted with one or more enzymaticallyacceptable groups. R^(A) may be the same or different from R^(B) instructure or chirality. R^(A) and R^(B), taken together, may form a ringthat is unsubstituted, substituted, or fused to other rings. Specificamino acceptor compounds are further described in the detaileddescription.

“Carbonyl by-product” and “keto by-product” refer to the carbonylcompound formed from the amino group donor when the amino group on theamino group donor is transferred to the amino group acceptor in atransamination reaction. The carbonyl by-product has the generalstructure of formula:

wherein R^(C) and R^(D) are defined above for the amino group donor.

“Pyridoxal-phosphate”, “PLP”, “pyridoxal-5′-phosphate”, “PYP”, and “P5P”are used interchangeably herein to refer to the compound that acts as acoenzyme in transaminase reactions. In some embodiments, pyridoxalphosphate is defined by the structure1-(4′-formyl-3′-hydroxy-2′-methyl-5′-pyridyl)methoxyphosphonic acid, CASnumber [54-47-7]. Pyridoxal-5′-phosphate is produced in vivo byphosphorylation and oxidation of pyridoxal (also known as pyridoxine orVitamin B6). In transamination reactions using transaminase enzymes, theamino group of the amino group donor is transferred to the coenzyme toproduce a keto byproduct, while pyridoxal-5′-phosphate is converted topyridoxamine phosphate. Pyridoxal-5′-phosphate is regenerated byreaction with a different keto compound (the amino group acceptor). Thetransfer of the amino group from pyridoxamine phosphate to the aminoacceptor produces a chiral amine and regenerates the coenzyme. Thepyridoxal-5′-phosphate of the current invention can be replaced by othermembers of the vitamin B₆ family, including, among others, pyridoxal(PL), pyridoxamine (PM), and their phosphorylated counterparts;pyridoxine phosphate (PNP), and pyridoxamine phosphate (PMP).

“Coding sequence” refers to that portion of a nucleic acid (e.g., agene) that encodes an amino acid sequence of a protein.

“Naturally occurring” or “wild-type” refers to the form found in nature.For example, a naturally occurring or wild-type polypeptide orpolynucleotide sequence is a sequence present in an organism that can beisolated from a source in nature and which has not been intentionallymodified by human manipulation.

“Recombinant” when used with reference to, e.g., a cell, nucleic acid,or polypeptide, refers to a material, or a material corresponding to thenatural or native form of the material, that has been modified in amanner that would not otherwise exist in nature, or is identical theretobut produced or derived from synthetic materials and/or by manipulationusing recombinant techniques. Non-limiting examples include, amongothers, recombinant cells expressing genes that are not found within thenative (non-recombinant) form of the cell or express native genes thatare otherwise expressed at a different level.

“Percentage of sequence identity,” “percent identity,” and “percentidentical” are used herein to refer to comparisons betweenpolynucleotide sequences or polypeptide sequences, and are determined bycomparing two optimally aligned sequences over a comparison window,wherein the portion of the polynucleotide or polypeptide sequence in thecomparison window may comprise additions or deletions (i.e., gaps) ascompared to the reference sequence for optimal alignment of the twosequences. The percentage is calculated by determining the number ofpositions at which either the identical nucleic acid base or amino acidresidue occurs in both sequences or a nucleic acid base or amino acidresidue is aligned with a gap to yield the number of matched positions,dividing the number of matched positions by the total number ofpositions in the window of comparison and multiplying the result by 100to yield the percentage of sequence identity. Determination of optimalalignment and percent sequence identity is performed using the BLAST andBLAST 2.0 algorithms (see e.g., Altschul et al., 1990, J. Mol. Biol.215: 403-410 and Altschul et al., 1977, Nucleic Acids Res. 3389-3402).Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information website.

Briefly, the BLAST analyses involve first identifying high scoringsequence pairs (HSPs) by identifying short words of length W in thequery sequence, which either match or satisfy some positive-valuedthreshold score T when aligned with a word of the same length in adatabase sequence. T is referred to as, the neighborhood word scorethreshold (Altschul et al, supra). These initial neighborhood word hitsact as seeds for initiating searches to find longer HSPs containingthem. The word hits are then extended in both directions along eachsequence for as far as the cumulative alignment score can be increased.Cumulative scores are calculated using, for nucleotide sequences, theparameters M (reward score for a pair of matching residues; always >0)and N (penalty score for mismatching residues; always <0). For aminoacid sequences, a scoring matrix is used to calculate the cumulativescore. Extension of the word hits in each direction are halted when: thecumulative alignment score falls off by the quantity X from its maximumachieved value; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, T,and X determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences) uses as defaults a wordlength (W) of11, an expectation (E) of 10, M=5, N=−4, and a comparison of bothstrands. For amino acid sequences, the BLASTP program uses as defaults awordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoringmatrix (see Henikoff and Henikoff, 1989, Proc Natl Acad Sci USA89:10915).

Numerous other algorithms are available that function similarly to BLASTin providing percent identity for two sequences. Optimal alignment ofsequences for comparison can be conducted, e.g., by the local homologyalgorithm of Smith and Waterman, 1981, Adv. Appl. Math. 2:482, by thehomology alignment algorithm of Needleman and Wunsch, 1970, J. Mol.Biol. 48:443, by the search for similarity method of Pearson and Lipman,1988, Proc. Natl. Acad. Sci. USA 85:2444, by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe GCG Wisconsin Software Package), or by visual inspection (seegenerally, Current Protocols in Molecular Biology, F. M. Ausubel et al.,eds., Current Protocols, a joint venture between Greene PublishingAssociates, Inc. and John Wiley & Sons, Inc., (1995 Supplement)(Ausubel)). Additionally, determination of sequence alignment andpercent sequence identity can employ the BESTFIT or GAP programs in theGCG Wisconsin Software package (Accelrys, Madison, Wis.), using defaultparameters provided.

“Reference sequence” refers to a defined sequence to which an alteredsequence is compared. A reference sequence may be a subset of a largersequence, for example, a segment of a full-length gene or polypeptidesequence. Generally, a reference sequence is at least 20 nucleotide oramino acid residues in length, at least 25 residues in length, at least50 residues in length, or the full length of the nucleic acid orpolypeptide. Since two polynucleotides or polypeptides may each (1)comprise a sequence (i.e., a portion of the complete sequence) that issimilar between the two sequences, and (2) may further comprise asequence that is divergent between the two sequences, sequencecomparisons between two (or more) polynucleotides or polypeptide aretypically performed by comparing sequences of the two polynucleotidesover a comparison window to identify and compare local regions ofsequence similarity.

The term “reference sequence” is not intended to be limited to wild-typesequences, and can include engineered or altered sequences. For example,in some embodiments, a “reference sequence” can be a previouslyengineered or altered amino acid sequence. For instance, a “referencesequence based on SEQ ID NO:2 having a glycine residue at position X284”refers to a reference sequence corresponding to SEQ ID NO:2 with aglycine residue at X284 (the un-altered version of SEQ ID NO:2 hasalanine at X284).

“Comparison window” refers to a conceptual segment of at least about 20contiguous nucleotide positions or amino acids residues wherein asequence may be compared to a reference sequence of at least 20contiguous nucleotides or amino acids and wherein the portion of thesequence in the comparison window may comprise additions or deletions(i.e., gaps) of 20 percent or less as compared to the reference sequence(which does not comprise additions or deletions) for optimal alignmentof the two sequences. The comparison window can be longer than 20contiguous residues, and includes, optionally 30, 40, 50, 100, or longerwindows.

“Substantial identity” refers to a polynucleotide or polypeptidesequence that has at least 80 percent sequence identity, at least 85percent sequence identity, at least 89 percent sequence identity, atleast 95 percent sequence identity, and even at least 99 percentsequence identity as compared to a reference sequence over a comparisonwindow of at least 20 residue positions, frequently over a window of atleast 30-50 residues, wherein the percentage of sequence identity iscalculated by comparing the reference sequence to a sequence thatincludes deletions or additions which total 20 percent or less of thereference sequence over the window of comparison. In specificembodiments applied to polypeptides, the term “substantial identity”means that two polypeptide sequences, when optimally aligned, such as bythe programs GAP or BESTFIT using default gap weights, share at least 80percent sequence identity, preferably at least 89 percent sequenceidentity, at least 95 percent sequence identity or more (e.g., 99percent sequence identity). Preferably, residue positions which are notidentical differ by conservative amino acid substitutions.

“Corresponding to”, “reference to” or “relative to” when used in thecontext of the numbering of a given amino acid or polynucleotidesequence refers to the numbering of the residues of a specifiedreference sequence when the given amino acid or polynucleotide sequenceis compared to the reference sequence. In other words, the residuenumber or residue position of a given polymer is designated with respectto the reference sequence rather than by the actual numerical positionof the residue within the given amino acid or polynucleotide sequence.For example, a given amino acid sequence, such as that of an engineeredtransaminase, can be aligned to a reference sequence by introducing gapsto optimize residue matches between the two sequences. In these cases,although the gaps are present, the numbering of the residue in the givenamino acid or polynucleotide sequence is made with respect to thereference sequence to which it has been aligned.

“Stereoselectivity” refers to the preferential formation in a chemicalor enzymatic reaction of one stereoisomer over another.Stereoselectivity can be partial, where the formation of onestereoisomer is favored over the other, or it may be complete where onlyone stereoisomer is formed. When the stereoisomers are enantiomers, thestereoselectivity is referred to as enantioselectivity, the fraction(typically reported as a percentage) of one enantiomer in the sum ofboth. It is commonly alternatively reported in the art (typically as apercentage) as the enantiomeric excess (e.e.) calculated therefromaccording to the formula [major enantiomer−minor enantiomer]/[majorenantiomer+minor enantiomer]. Where the stereoisomers arediastereoisomers, the stereo selectivity is referred to asdiastereoselectivity, the fraction (typically reported as a percentage)of one diastereomer in a mixture of two diastereomers, commonlyalternatively reported as the diastereomeric excess (d.e.). Enantiomericexcess and diastereomeric excess are types of stereomeric excess.

“Highly stereoselective” refers to a chemical or enzymatic reaction thatis capable of converting a substrate (e.g., formula (II)) to itscorresponding product (e.g., formula (I)) with at least about 85%stereoisomeric excess.

“Improved enzyme property” refers to any enzyme property made better ormore desirable for a particular purpose as compared to that propertyfound in a reference enzyme. For the engineered transaminasepolypeptides described herein, the comparison is generally made to thewild-type transaminase enzyme, although in some embodiments, thereference transaminase can be another improved engineered transaminase.Enzyme properties for which improvement can be made include, but are notlimited to, enzymatic activity (which can be expressed in terms ofpercent conversion of the substrate in a period of time), thermalstability, solvent stability, pH activity profile, coenzymerequirements, refractoriness to inhibitors (e.g., product inhibition),stereospecificity, and stereoselectivity (including enantioselectivity).

“Increased enzymatic activity” or “increased activity” refers to animproved property of an engineered enzyme, which can be represented byan increase in specific activity (e.g., product produced/time/weightprotein) or an increase in percent conversion of the substrate to theproduct (e.g., percent conversion of starting amount of substrate toproduct in a specified time period using a specified amount oftransaminase) as compared to a reference enzyme. Exemplary methods todetermine enzyme activity are provided in the Examples. Any propertyrelating to enzyme activity may be affected, including the classicalenzyme properties of K_(m), V_(max), or k_(act), changes of which canlead to increased enzymatic activity. Improvements in enzyme activitycan be from about 1.5 times the enzymatic activity of the correspondingwild-type or engineered enzyme, to as much as 2 times, 5 times, 10times, 20 times, 25 times, 50 times, 75 times, 100 times, or moreenzymatic activity than the naturally occurring enzyme (e.g., atransaminase) or another engineered enzyme from which the enzymesexhibiting increased activity were derived. In specific embodiments, theengineered transaminase enzymes of the present disclosure exhibitimproved enzymatic activity in the range of 1.5 to 50 times, 1.5 to 100times or greater than that of the parent transaminase enzyme (i.e., thewild-type or engineered transaminase from which they were derived). Itis understood by the skilled artisan that the activity of any enzyme isdiffusion limited such that the catalytic turnover rate cannot exceedthe diffusion rate of the substrate, including any required coenzymes.The theoretical maximum of the diffusion limit is generally about 10⁸ to10⁹ (M⁻¹s⁻¹). Hence, any improvements in the enzyme activity of thetransaminase will have an upper limit related to the diffusion rate ofthe substrates acted on by the transaminase enzyme. Transaminaseactivity can be measured by any one of standard assays used formeasuring transaminases, such as change in substrate or productconcentration, or change in concentration of the amino group donor.Comparisons of enzyme activities are made using a defined preparation ofenzyme, a defined assay under a set condition, and one or more definedsubstrates, as further described in detail herein. Generally, whenenzymes in cell lysates are compared, the numbers of cells and theamount of protein assayed are determined as well as use of identicalexpression systems and identical host cells to minimize variations inamount of enzyme produced by the host cells and present in the lysates.

“Conversion” refers to the enzymatic transformation of a substrate tothe corresponding product. “Percent conversion” refers to the percent ofthe substrate that is converted to the product within a period of timeunder specified conditions. Thus, for example, the “enzymatic activity”or “activity” of a transaminase polypeptide can be expressed as “percentconversion” of the substrate to the product.

“Thermostable” or “thermal stable” are used interchangeably to refer toa polypeptide that is resistant to inactivation when exposed to a set oftemperature conditions (e.g., 40-80° C.) for a period of time (e.g.,0.5-24 hrs) compared to the untreated enzyme, thus retaining a certainlevel of residual activity (more than 60% to 80% for example) afterexposure to elevated temperatures.

“Solvent stable” refers to a polypeptide that maintains similar activity(more than e.g., 60% to 80%) after exposure to varying concentrations(e.g., 5-99%) of solvent , (e.g., isopropyl alcohol, dimethylsulfoxide,tetrahydrofuran, 2-methyltetrahydrofuran, acetone, toluene,butylacetate, methyl tert-butylether, acetonitrile, etc.) for a periodof time (e.g., 0.5-24 hrs) compared to the untreated enzyme.

“pH stable” refers to a polypeptide that maintains similar activity(more than e.g., 60% to 80%) after exposure to low or high pH (e.g.,4.5-6 or 8 to 12) for a period of time (e.g., 0.5-24 hrs) compared tothe untreated enzyme.

“Thermo- and solvent stable” refers to a polypeptide that is boththermostable and solvent stable.

“Derived from” as used herein in the context of engineered enzymesidentifies the originating enzyme, and/or the gene encoding such enzyme,upon which the engineering was based. For example, the engineeredtransaminase enzyme of SEQ ID NO: 4 was obtained by mutating thetransaminase of SEQ ID NO:2. Thus, this engineered transaminase enzymeof SEQ ID NO:4 is “derived from” the type polypeptide of SEQ ID NO:2.

“Amino acid” or “residue” as used in context of the polypeptidesdisclosed herein refers to the specific monomer at a sequence position(e.g., P8 indicates that the “amino acid” or “residue” at position 8 ofSEQ ID NO: 2 is a proline.)

“Hydrophilic amino acid or residue” refers to an amino acid or residuehaving a side chain exhibiting a hydrophobicity of less than zeroaccording to the normalized consensus hydrophobicity scale of Eisenberget al., 1984, J. Mol. Biol. 179:125-142. Genetically encoded hydrophilicamino acids include L-Thr (T), L-Ser (S), L-His (H), L-Glu (E), L-Asn(N), L-Gln (Q), L-Asp (D), L-Lys (K) and L-Arg (R).

“Acidic amino acid or residue” refers to a hydrophilic amino acid orresidue having a side chain exhibiting a pK value of less than about 6when the amino acid is included in a peptide or polypeptide. Acidicamino acids typically have negatively charged side chains atphysiological pH due to loss of a hydrogen ion. Genetically encodedacidic amino acids include L-Glu (E) and L-Asp (D).

“Basic amino acid or residue” refers to a hydrophilic amino acid orresidue having a side chain exhibiting a pKa value of greater than about6 when the amino acid is included in a peptide or polypeptide. Basicamino acids typically have positively charged side chains atphysiological pH due to association with hydronium ion. Geneticallyencoded basic amino acids include L-Arg (R) and L-Lys (K).

“Polar amino acid or residue” refers to a hydrophilic amino acid orresidue having a side chain that is uncharged at physiological pH, butwhich has at least one bond in which the pair of electrons shared incommon by two atoms is held more closely by one of the atoms.Genetically encoded polar amino acids include L-Asn (N), L-Gln (Q),L-Ser (S) and L-Thr (T).

“Hydrophobic amino acid or residue” refers to an amino acid or residuehaving a side chain exhibiting a hydrophobicity of greater than zeroaccording to the normalized consensus hydrophobicity scale of Eisenberget al., 1984, J. Mol. Biol. 179:125-142. Genetically encoded hydrophobicamino acids include L-Pro (P), L-Ile (I), L-Phe (F), L-Val (V), L-Leu(L), L-Trp (W), L-Met (M), L-Ala (A) and L-Tyr (Y).

“Aromatic amino acid or residue” refers to a hydrophilic or hydrophobicamino acid or residue having a side chain that includes at least onearomatic or heteroaromatic ring. Genetically encoded aromatic aminoacids include L-Phe (F), L-Tyr (Y) and L-Trp (W). Although owing to thepKa of its heteroaromatic nitrogen atom L-His (H) it is sometimesclassified as a basic residue, or as an aromatic residue as its sidechain includes a heteroaromatic ring, herein histidine is classified asa hydrophilic residue or as a “constrained residue” (see below).

“Constrained amino acid or residue” refers to an amino acid or residuethat has a constrained geometry. Herein, constrained residues includeL-Pro (P) and L-His (H). Histidine has a constrained geometry because ithas a relatively small imidazole ring. Proline has a constrainedgeometry because it also has a five membered ring.

“Non-polar amino acid or residue” refers to a hydrophobic amino acid orresidue having a side chain that is uncharged at physiological pH andwhich has bonds in which the pair of electrons shared in common by twoatoms is generally held equally by each of the two atoms (i.e., the sidechain is not polar). Genetically encoded non-polar amino acids includeL-Gly (G), L-Leu (L), L-Val (V), L-Ile (I), L-Met (M) and L-Ala (A).

“Aliphatic amino acid or residue” refers to a hydrophobic amino acid orresidue having an aliphatic hydrocarbon side chain. Genetically encodedaliphatic amino acids include L-Ala (A), L-Val (V), L-Leu (L) and L-Ile(I).

“Cysteine” or L-Cys (C) is unusual in that it can form disulfide bridgeswith other L-Cys (C) amino acids or other sulfanyl- orsulfhydryl-containing amino acids. The “cysteine-like residues” includecysteine and other amino acids that contain sulfhydryl moieties that areavailable for formation of disulfide bridges. The ability of L-Cys (C)(and other amino acids with —SH containing side chains) to exist in apeptide in either the reduced free —SH or oxidized disulfide-bridgedform affects whether L-Cys (C) contributes net hydrophobic orhydrophilic character to a peptide. While L-Cys (C) exhibits ahydrophobicity of 0.29 according to the normalized consensus scale ofEisenberg (Eisenberg et al., 1984, supra), it is to be understood thatfor purposes of the present disclosure L-Cys (C) is categorized into itsown unique group.

“Small amino acid or residue” refers to an amino acid or residue havinga side chain that is composed of a total of three or fewer carbon and/orheteroatoms (excluding the α-carbon and hydrogens). The small aminoacids or residues may be further categorized as aliphatic, non-polar,polar or acidic small amino acids or residues, in accordance with theabove definitions. Genetically-encoded small amino acids include L-Ala(A), L-Val (V), L-Cys (C), L-Asn (N), L-Ser (S), L-Thr (T) and L-Asp(D).

“Hydroxyl-containing amino acid or residue” refers to an amino acidcontaining a hydroxyl (—OH) moiety. Genetically-encodedhydroxyl-containing amino acids include L-Ser (S) L-Thr (T) and L-Tyr(Y).

“Amino acid difference” or “residue difference” refers to a change inthe residue at a specified position of a polypeptide sequence whencompared to a reference sequence. For example, a residue difference atposition X8, where the reference sequence has a serine, refers to achange of the residue at position X8 to any residue other than serine.As disclosed herein, an enzyme can include one or more residuedifferences relative to a reference sequence, where multiple residuedifferences typically are indicated by a list of the specified positionswhere changes are made relative to the reference sequence (e.g., “one ormore residue differences as compared to SEQ ID NO:2 at the followingresidue positions: X4; X5; X8; X18; X25; X26; X27; X28; X30; X41; X42;X48; X49; X50; X54; X55; X60; X61; X62; X65; X81; X94; X96; X102; X117;X120; X124; X126; X136; X137; X138; X146; X148; X150; X152; X155; X156;X160; X163; X164; X169; X174; X178; X195; X199; X204; X208; X209; X211;X215; X217; X225; X230; X252; X269; X273; X282; X292; X297; X302; X306;X321; and X329.”).

“Conservative” amino acid substitutions or mutations refer to theinterchangeability of residues having similar side chains, and thustypically involves substitution of the amino acid in the polypeptidewith amino acids within the same or similar defined class of aminoacids. However, as used herein, in some embodiments, conservativemutations do not include substitutions from a hydrophilic tohydrophilic, hydrophobic to hydrophobic, hydroxyl-containing tohydroxyl-containing, or small to small residue, if the conservativemutation can instead be a substitution from an aliphatic to analiphatic, non-polar to non-polar, polar to polar, acidic to acidic,basic to basic, aromatic to aromatic, or constrained to constrainedresidue. Further, as used herein, A, V, L, or I can be conservativelymutated to either another aliphatic residue or to another non-polarresidue. The table below shows exemplary conservative substitutions.

TABLE 1 Residue Possible Conservative Mutations A, L, V, I Otheraliphatic (A, L, V, I) Other non-polar (A, L, V, I, G, M) G, M Othernon-polar (A, L, V, I, G, M) D, E Other acidic (D, E) K, R Other basic(K, R) P, H Other constrained (P, H) N, Q, S, T Other polar Y, W, FOther aromatic (Y, W, F) C None

“Non-conservative substitution” refers to substitution or mutation of anamino acid in the polypeptide with an amino acid with significantlydiffering side chain properties. Non-conservative substitutions may useamino acids between, rather than within, the defined groups listedabove. In one embodiment, a non-conservative mutation affects (a) thestructure of the peptide backbone in the area of the substitution (e.g.,proline for glycine) (b) the charge or hydrophobicity, or (c) the bulkof the side chain.

“Deletion” refers to modification of the polypeptide by removal of oneor more amino acids from the reference polypeptide. Deletions cancomprise removal of 1 or more amino acids, 2 or more amino acids, 5 ormore amino acids, 10 or more amino acids, 15 or more amino acids, or 20or more amino acids, up to 10% of the total number of amino acids, up to20% of the total number of amino acids, or up to 30% of the total numberof amino acids making up the polypeptide while retaining enzymaticactivity and/or retaining the improved properties of an engineeredtransaminase enzyme. Deletions can be directed to the internal portionsand/or terminal portions of the polypeptide. In various embodiments, thedeletion can comprise a continuous segment or can be discontinuous.

“Insertion” refers to modification of the polypeptide by addition of oneor more amino acids to the reference polypeptide. In some embodiments,the improved engineered transaminase enzymes comprise insertions of oneor more amino acids to the naturally occurring transaminase polypeptideas well as insertions of one or more amino acids to other improvedtransaminase polypeptides. Insertions can be in the internal portions ofthe polypeptide, or to the carboxy or amino terminus. Insertions as usedherein include fusion proteins as is known in the art. The insertion canbe a contiguous segment of amino acids or separated by one or more ofthe amino acids in the naturally occurring polypeptide.

“Fragment” as used herein refers to a polypeptide that has anamino-terminal and/or carboxy-terminal deletion, but where the remainingamino acid sequence is identical to the corresponding positions in thesequence. Fragments can be at least 14 amino acids long, at least 20amino acids long, at least 50 amino acids long or longer, and up to 70%,80%, 90%, 95%, 98%, and 99% of the full-length transaminase polypeptide,for example the polypeptide of SEQ ID NO:4.

“Isolated polypeptide” refers to a polypeptide which is substantiallyseparated from other contaminants that naturally accompany it, e.g.,protein, lipids, and polynucleotides. The term embraces polypeptideswhich have been removed or purified from their naturally-occurringenvironment or expression system (e.g., host cell or in vitrosynthesis). The improved transaminase enzymes may be present within acell, present in the cellular medium, or prepared in various forms, suchas lysates or isolated preparations. As such, in some embodiments, theimproved transaminase enzyme can be an isolated polypeptide.

“Substantially pure polypeptide” refers to a composition in which thepolypeptide species is the predominant species present (i.e., on a molaror weight basis it is more abundant than any other individualmacromolecular species in the composition), and is generally asubstantially purified composition when the object species comprises atleast about 50 percent of the macromolecular species present by mole or% weight. Generally, a substantially pure transaminase composition willcomprise about 60% or more, about 70% or more, about 80% or more, about90% or more, about 95% or more, and about 98% or more of allmacromolecular species by mole or % weight present in the composition.In some embodiments, the object species is purified to essentialhomogeneity (i.e., contaminant species cannot be detected in thecomposition by conventional detection methods) wherein the compositionconsists essentially of a single macromolecular species. Solventspecies, small molecules (<500 Daltons), and elemental ion species arenot considered macromolecular species. In some embodiments, the isolatedimproved transaminases polypeptide is a substantially pure polypeptidecomposition.

“Stringent hybridization” is used herein to refer to conditions underwhich nucleic acid hybrids are stable. As known to those of skill in theart, the stability of hybrids is reflected in the melting temperature(T_(m)) of the hybrids. In general, the stability of a hybrid is afunction of ion strength, temperature, G/C content, and the presence ofchaotropic agents. The T_(m) values for polynucleotides can becalculated using known methods for predicting melting temperatures (see,e.g., Baldino et al., Methods Enzymology 168:761-777; Bolton et al.,1962, Proc. Natl. Acad. Sci. USA 48:1390; Bresslauer et al., 1986, Proc.Natl. Acad. Sci USA 83:8893-8897; Freier et al., 1986, Proc. Natl. Acad.Sci USA 83:9373-9377; Kierzek et al., Biochemistry 25:7840-7846; Rychliket al., 1990, Nucleic Acids Res 18:6409-6412 (erratum, 1991, NucleicAcids Res 19:698); Sambrook et al., supra); Suggs et al., 1981, InDevelopmental Biology Using Purified Genes (Brown et al., eds.), pp.683-693, Academic Press; and Wetmur, 1991, Crit Rev Biochem Mol Biol26:227-259. All publications incorporate herein by reference). In someembodiments, the polynucleotide encodes the polypeptide disclosed hereinand hybridizes under defined conditions, such as moderately stringent orhighly stringent conditions, to the complement of a sequence encoding anengineered transaminase enzyme of the present disclosure.

“Hybridization stringency” relates to hybridization conditions, such aswashing conditions, in the hybridization of nucleic acids. Generally,hybridization reactions are performed under conditions of lowerstringency, followed by washes of varying but higher stringency. Theterm “moderately stringent hybridization” refers to conditions thatpermit target-DNA to bind a complementary nucleic acid that has about60% identity, preferably about 75% identity, about 85% identity to thetarget DNA; with greater than about 90% identity totarget-polynucleotide. Exemplary moderately stringent conditions areconditions equivalent to hybridization in 50% formamide, 5× Denhart'ssolution, 5× SSPE, 0.2% SDS at 42° C., followed by washing in 0.2× SSPE,0.2% SDS, at 42° C. “High stringency hybridization” refers generally toconditions that are about 10° C. or less from the thermal meltingtemperature T_(m) as determined under the solution condition for adefined polynucleotide sequence. In some embodiments, a high stringencycondition refers to conditions that permit hybridization of only thosenucleic acid sequences that form stable hybrids in 0.018M NaCl at 65° C.(i.e., if a hybrid is not stable in 0.018M NaCl at 65° C., it will notbe stable under high stringency conditions, as contemplated herein).High stringency conditions can be provided, for example, byhybridization in conditions equivalent to 50% formamide, 5× Denhart'ssolution, 5× SSPE, 0.2% SDS at 42° C., followed by washing in 0.1× SSPE,and 0.1% SDS at 65° C. Another high stringency condition is hybridizingin conditions equivalent to hybridizing in 5× SSC containing 0.1% (w:v)SDS at 65° C. and washing in 0.1× SSC containing 0.1% SDS at 65° C.Other high stringency hybridization conditions, as well as moderatelystringent conditions, are described in the references cited above.

“Heterologous” polynucleotide refers to any polynucleotide that isintroduced into a host cell by laboratory techniques, and includespolynucleotides that are removed from a host cell, subjected tolaboratory manipulation, and then reintroduced into a host cell.

“Codon optimized” refers to changes in the codons of the polynucleotideencoding a protein to those preferentially used in a particular organismsuch that the encoded protein is efficiently expressed in the organismof interest. Although the genetic code is degenerate in that most aminoacids are represented by several codons, called “synonyms” or“synonymous” codons, it is well known that codon usage by particularorganisms is nonrandom and biased towards particular codon triplets.This codon usage bias may be higher in reference to a given gene, genesof common function or ancestral origin, highly expressed proteins versuslow copy number proteins, and the aggregate protein coding regions of anorganism's genome. In some embodiments, the polynucleotides encoding thetransaminases enzymes may be codon optimized for optimal production fromthe host organism selected for expression.

“Preferred, optimal, high codon usage bias codons” refersinterchangeably to codons that are used at higher frequency in theprotein coding regions than other codons that code for the same aminoacid. The preferred codons may be determined in relation to codon usagein a single gene, a set of genes of common function or origin, highlyexpressed genes, the codon frequency in the aggregate protein codingregions of the whole organism, codon frequency in the aggregate proteincoding regions of related organisms, or combinations thereof. Codonswhose frequency increases with the level of gene expression aretypically optimal codons for expression. A variety of methods are knownfor determining the codon frequency (e.g., codon usage, relativesynonymous codon usage) and codon preference in specific organisms,including multivariate analysis, for example, using cluster analysis orcorrespondence analysis, and the effective number of codons used in agene (see GCG CodonPreference, Genetics Computer Group WisconsinPackage; CodonW, John Peden, University of Nottingham; McInerney, J. O,1998, Bioinformatics 14:372-73; Stenico et al., 1994, Nucleic Acids Res.222437-46; Wright, F., 1990, Gene 87:23-29). Codon usage tables areavailable for a growing list of organisms (see for example, Wada et al.,1992, Nucleic Acids Res. 20:2111-2118; Nakamura et al., 2000, Nucl.Acids Res. 28:292; Duret, et al., supra; Henaut and Danchin,“Escherichia coli and Salmonella,” 1996, Neidhardt, et al. Eds., ASMPress, Washington D.C., p. 2047-2066. The data source for obtainingcodon usage may rely on any available nucleotide sequence capable ofcoding for a protein. These data sets include nucleic acid sequencesactually known to encode expressed proteins (e.g., complete proteincoding sequences-CDS), expressed sequence tags (EST), or predictedcoding regions of genomic sequences (see for example, Mount, D.,Bioinformatics: Sequence and Genome Analysis, Chapter 8, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 2001; Uberbacher, E.C., 1996, Methods Enzymol. 266:259-281; Tiwari et al., 1997, Comput.Appl. Biosci. 13:263-270).

“Control sequence” is defined herein to include all components, whichare necessary or advantageous for the expression of a polynucleotideand/or polypeptide of the present disclosure. Each control sequence maybe native or foreign to the polynucleotide of interest. Such controlsequences include, but are not limited to, a leader, polyadenylationsequence, propeptide sequence, promoter, signal peptide sequence, andtranscription terminator.

“Operably linked” is defined herein as a configuration in which acontrol sequence is appropriately placed (i.e., in a functionalrelationship) at a position relative to a polynucleotide of interestsuch that the control sequence directs or regulates the expression ofthe polynucleotide and/or polypeptide of interest.

“Promoter sequence” is a nucleic acid sequence that is recognized by ahost cell for expression of a polynucleotide of interest, such as acoding sequence. The control sequence may comprise an appropriatepromoter sequence. The promoter sequence contains transcriptionalcontrol sequences, which mediate the expression of a polynucleotide ofinterest. The promoter may be any nucleic acid sequence which showstranscriptional activity in the host cell of choice including mutant,truncated, and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

“Alkyl” refers to straight or branched chain hydrocarbon groups. Whennumbers appear in subscript after the symbol “C”, the subscript defineswith more specificity the number of carbon atoms that a particular groupcan contain. For example, “C₁-C₆ alkyl” refers to straight and branchedchain alkyl groups with one to six carbon atoms, such as methyl, ethyl,n-propyl, isopropyl, n-butyl, t-butyl, n-pentyl, and so forth. Alkylgroups may be optionally substituted with one or more substituentgroups. As used herein, alkyl includes alkanyl and alkylene, as furtherdescribed below.

“Alkanyl” by itself or as part of another substituent refers to asaturated branched, straight-chain or cyclic alkyl derived by theremoval of one hydrogen atom from a single carbon atom of a parentalkane. Alkanyl groups include, but are not limited to methanyl;ethanyl; propanyls such as propan-1-yl, propan-2-yl (isopropyl),cyclopropan-1-yl, etc.; butanyls such as butan-1-yl, butan-2-yl(sec-butyl), 2-methyl-propan-1-yl(isobutyl), 2-methyl-propan-2-yl(t-butyl), cyclobutan-1-yl, etc.; and the like. In some embodiments, thealkanyl groups are (C₁-C₆) alkanyl.

“Alkylene” by itself or as part of another substituent refers to asaturated or unsaturated, branched, straight-chain or cyclic divalenthydrocarbon radical derived by the removal of two hydrogen atoms from asingle carbon atom or two different carbon atoms of a parent alkane,alkene or alkyne. For example, —CH₂CH₃ is an ethyl, while —CH₂CH₂— is anethylene. The term “alkylene” includes “cycloalkylene”. The term“alkylene” is specifically intended to include groups having any degreeor level of saturation, i.e., groups having exclusively singlecarbon-carbon bonds, groups having one or more double carbon-carbonbonds, groups having one or more triple carbon-carbon bonds and groupshaving mixtures of single, double and triple carbon-carbon bonds.

“Alkenyl” and “alkene” refer to straight or branched chain hydrocarbongroups having at least one double bond. When numbers appear in subscriptafter the symbol “C”, the subscript defines with more specificity thenumber of carbon atoms that a particular group can contain. For example,“C₁-C₆ alkenyl” refers to straight and branched chain alkyl groups withone to six carbon atoms that include at least one double bond. Typicalalkenyl groups include are well known in the art, and include, but arenot limited to: ethenyl, 1-methyl-ethenyl, 1- or 2-propenyl,1-methyl-1-propenyl, 1-methyl-2-propenyl, 1,1-dimethyl-2-propenyl,2-methyl-2-propenyl, 1-, 2- or 3-butenyl, 1-methyl-1-butenyl,2-methyl-1-butenyl, 3-methyl-1-butenyl, 3,3-dimethyl-1-butenyl,2,3-dimethyl-1-butenyl, 1-methyl-2-butenyl, 1,1-dimethyl-2-butenyl,2-methyl-2-butenyl, 3-methyl-2-butenyl, 1,3-butadienyl,1,3-dimethyl-1,3-butadienyl, 1-, 2-, 3- or 4-pentenyl, and so forth.Alkenyl groups may be optionally substituted with one or moresubstituent groups.

“Alkynyl” and “alkyne” refer to straight or branched chain hydrocarbongroups having at least one triple bond. When numbers appear in subscriptafter the symbol “C”, the subscript defines with more specificity thenumber of carbon atoms that a particular group can contain. For example,“C₁-C₆ alkynyl” refers to straight and branched chain alkyl groups withone to six carbon atoms that include at least one triple bond. Typicalalkynyl groups include are well known in the art, and include, but arenot limited to: ethynyl, 1- or 2-propynyl, 1-methyl-2-propynyl,1,1-dimethyl-2-propynyl, 1-, 2- or 3-butynyl, 3-methyl-1-butynyl,3,3-dimethyl-1-butynyl, 1-methyl-2butynyl, 1,1-dimethyl-2-butynyl, 1-,2-, 3-, or 4-pentynyl, and so forth. Alkynyl groups may be optionallysubstituted with one or more substituent groups.

“Aryl” refers to a monovalent aromatic hydrocarbon radical of 6 to about20 carbon atoms derived by the removal of one hydrogen atom from asingle carbon atom of a parent aromatic ring system. Aryl groupsinclude, but are not limited to, groups derived from aceanthrylene,acenaphthylene, acephenanthrylene, anthracene, azulene, benzene,chrysene, coronene, fluoranthene, fluorene, hexacene, hexaphene,hexalene, as indacene, s-indacene, indane, indene, naphthalene,octacene, octaphene, octalene, ovalene, penta-2,4-diene, pentacene,pentalene, pentaphene, perylene, phenalene, phenanthrene, picene,pleiadene, pyrene, pyranthrene, rubicene, triphenylene, trinaphthalene,and the like, as well as the various hydro isomers thereof. In someembodiments, the aryl group is (C₅-C₁₅) aryl, with (C₅-C₁₀) beingpreferred. In some embodiments, the aryls are cyclopentadienyl, phenyland naphthyl. Aryl groups may be optionally substituted with one or moresubstituent groups.

“Heteroaryl” and “heteroaromatic” refer to an aryl group in which one ormore of the carbon atoms of the parent aromatic ring system are replacedby a heteroatom (O, N, or S). Typical heteroaryl groups include, but arenot limited to, groups derived from acridine, benzimidazole,benzisoxazole, benzodioxan, benzodiaxole, benzofuran, benzopyrone,benzothiadiazole, benzothiazole, benzotriazole, benzoxazine,benzoxazole, benzoxazoline, carbazole, β-carboline, chromane, chromene,cinnoline, furan, imidazole, indazole, indole, indoline, indolizine,isobenzofuran, isochromene, isoindole, isoindoline, isoquinoline,isothiazole, isoxazole, naphthyridine, oxadiazole, oxazole, perimidine,phenanthridine, phenanthroline, phenazine, phthalazine, pteridine,purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine,pyrrole, pyrrolizine, quinazoline, quinoline, quinolizine, quinoxaline,tetrazole, thiadiazole, thiazole, thiophene, triazole, xanthene, and thelike, as well as the various hydro isomers thereof. In some embodiments,the heteroaryl group is a 5-14 membered heteroaryl. In some embodiments,the heteroaryl group is a 5-10 membered heteroaryl.

“Acyl” by itself or as part of another substituent refers to —C(O)R^(a),where R^(a) is hydrogen, or substituted or unsubstituted alkyl,cylcoalkyl, cycloheteroalkyl, aryl, arylalkyl, heteroalkyl, heteroaryl,or heteroarylalkyl as defined herein. Typical acyl groups include, butare not limited to, formyl, acetyl, cyclohexylcarbonyl,cyclohexylmethylcarbonyl, benzoyl, benzylcarbonyl, and the like.

“Acyloxy” by itself or as part of another substituent refers to—OC(O)R^(b), where R^(b) represents a hydrogen, or substituted orunsubstituted alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl,arylalkyl, and heteroaryl groups as defined herein. Alkylacyloxy refersan acyloxy where R^(b) is (C₁-C₁₂) alkyl, (C₁-C₈) alkyl, or (C₁-C₄)alkyl. Arylacyloxy refers to an acyloxy where R^(b) is an aryloptionally substituted with selected substituents, including, but notlimited to, hydroxyl, alkyl, halogen, (C₁-C₄) alkyl, (C₁-C₄) alkoxy, andcarboxyl.

“Alkoxy” by itself or as part of another substituent refers to —OR^(c),where R^(c) represents an alkyl or cycloalkyl group as defined herein.Typical alkoxy groups include, but are not limited to, methoxy, ethoxy,propoxy, butoxy, cyclohexyloxy, and the like.

“Alkylcarbonyl” by itself or as part of another substituent refers to—C(O)—R^(d′), where R^(d′) is an alkyl, as defined above. Typicalalkoxycarbonyl include, but are not limited to, acetyl, ethylcarbonyl,n-propylcarbonyl, and the like.

“Alkylthio” by itself or as part of another substituent, refers to—S—R^(e). where R^(e) is an alkyl. Typical alkylthio include, but arenot limited to, methylthio, ethylthio, n-propylthio, and the like.

“Alkoxycarbonyl” by itself or as part of another substituent, refers toC(O)OR^(f) where R^(f) represents an alkyl or cycloalkyl group asdefined herein. Typical alkoxycarbonyl groups include, but are notlimited to, methoxycarbonyl, ethoxycarbonyl, proproxycarbonyl,butoxycarbonyl, cyclohexyloxycarbonyl, and the like.

“Alkoxycarbonylalkyl” by itself or as part of another substituent refersto —R^(g)—C(O)OR^(h), where each R^(g) and R^(h) is independently analkyl. Typical alkoxycarbonylalkyl include, but are not limited to,methoxycarbonylmethyl, (1,1-dimethylethoxy)carbonylmethyl,2-(methoxycarbonyl)ethyl, and the like.

“Amino” by itself or as part of another substituent refers to the group—NH₂. Substituted amino refers to the group —NHR^(i′), NR^(i)R^(j′), andNR^(i)R^(j)R^(k), where each R^(i), R^(j), and R^(k) are independentlyselected from substituted or unsubstituted alkyl, cycloalkyl,cycloheteroalkyl, alkoxy, aryl, heteroaryl, heteroarylalkyl, acyl,alkoxycarbonyl, sulfanyl, sulfinyl, sulfonyl, and the like. Typicalamino groups include, but are limited to, dimethylamino, diethylamino,trimethylamino, triethylamino, methylysulfonylamino,furanyl-oxy-sulfamino, and the like.

“Carbonyl” refers to —C(═O).

“Halogen” or “Halo” by themselves or as part of another substituent,unless otherwise stated, refer to fluoro, chloro, bromo and iodo.

“Haloalkyl” by itself or as part of another substituent refers to analkyl group in which one or more of the hydrogen atoms is replaced witha halogen. Thus, the term “haloalkyl” is meant to includemonohaloalkyls, dihaloalkyls, trihaloalkyls, etc. up to perhaloalkyls.For example, the expression “(C₁-C₂) haloalkyl” includes 1-fluoromethyl,difluoromethyl, trifluoromethyl, 1-fluoroethyl, 1,1-difluoroethyl,1,2-difluoroethyl, 1,1,1-trifluoroethyl, perfluoroethyl, etc.

“Substituted” when used to modify a specified group or radical, meansthat one or more hydrogen atoms of the specified group or radical areeach, independently of one another, replaced with the same or differentsubstituent(s). Typical substituents (also referred to herein as“substituents”, “substituent groups,” “functional groups,” or “groups”)are well known in the art and include, but are not limited toheteroatoms or halo groups (e.g., —F, —Cl, —Br, —I), straight-chain,branched, or cyclic alkyls, straight-chain, branched, or cyclicalkenyls, heteroatom substituted alkyls or alkenyls (e.g., —O-alkyl,—S-alkyl), aryl or heteroaryl, and other functional groups with orwithout heteroatoms (e.g., —OH, —NH₂, —CF₃, —CN, —OCN, —SCN, —NO, and—NO₂). When a first substituent group is “substituted with one or more”second groups, one or more hydrogen atoms of the first group arereplaced with a corresponding number of second groups. When the numberof second groups is two or greater, each second group can be the same ordifferent.

“Substituted alkyl, aryl, or heteroaryl” refers to an alkyl, aryl, orheteroaryl group in which one or more hydrogen atoms is replaced withanother substituent group. Exemplary substituent groups include, but arenot limited to, —OR^(l), —SR^(l), —NR^(l)R^(m), —NO₂, —NO, —CN, —CF₃,halogen (e.g., —F, —Cl, —Br and —I), —C(O)R^(l), —C(O)OR^(l),—C(O)NR^(l), —S(O)₂R^(l), —S(O)₂NR^(l)R^(m), where each R^(l) and R^(m)are independently selected from the group consisting of hydrogen and(C₁-C₄) alkyl.

“Optional” or “optionally” refers to a described event or circumstancemay or may not occur, and that the description includes instances wherethe event or circumstance occurs and instances where the event orcircumstance does not. For example, “optionally substituted aryl” refersto an aryl group that may or may not be substituted and that thedescription encompasses both substituted aryl group and unsubstitutedaryl group.

“Stereoisomer,” “stereoisomeric form,” and the like are usedinterchangeably herein to refer to all isomers of individual moleculesthat differ only in the orientation of their atoms in space. In includesenantiomers and isomers of compounds with more than one chiral centerthat are not mirror images of one another (“diastereomers”).

“Chiral center” refers to a carbon atom to which four different groupsare attached.

“Enantiomer” or “enantiomeric” refers to a molecule that isnonsuperimposable on its mirror image and hence optically active wherethe enantiomer rotates the plane of polarized light in one direction andits mirror image rotates the plane of polarized light in the oppositedirection.

“Enriched” in reference to component in a composition, such as aparticular chiral compound, enantiomer, or diastereomer refers to acomposition where the component comprises greater than 50% and typicallycomprises at least about 60%, 70%, 80%, 90%, or even more of thatparticular chiral compound, enantiomer, or diastereomer. The amount ofenrichment can be determined using conventional analytical methodsroutinely used by those of ordinary skill in the art, including but notlimited to, NMR spectroscopy in the presence of chiral shift reagents,gas chromatographic analysis using chiral columns, and high pressureliquid chromatographic analysis using chiral columns. In someembodiments a single chiral compound, enantiomer, or diastereomer willbe substantially free of other corresponding chiral compound,enantiomer, or diastereomers. Chirally enantiomerically, ordiastereomerically enriched compositions that contain at least about 95%of a specified chiral compound, enantiomer, or diastereomer are referredto herein as “substantially chirally pure,” “substantiallyenantiomerically pure” and “substantially diastereomerically pure,”respectively. Compositions that contain at least about 99% of aspecified chiral compound, enantiomer, or diastereomer are referred toherein as “chirally pure,” “enantiomerically pure,” and“diastereomerically pure,” respectively.

“Compound” as used herein refers to any compounds encompassed by theidentified structural formula and/or chemical name associated with thecompound as disclosed herein. Compounds may be identified either bytheir chemical structure and/or chemical name. When the chemicalstructure and chemical name conflict, the chemical structure isdeterminative of the identity of the compound. Each compound identifiedby structural formulae and/or chemical name disclosed herein may containone or more chiral centers and/or double bonds, and therefore, may existas more than one stereoisomer, such as double-bond isomers (i.e.,geometric isomers), enantiomer, or diastereomer. Accordingly, unlessspecifically depicted or noted otherwise, the chemical structuresdepicted herein encompass all possible enantiomers and stereoisomers ofthe illustrated compounds including the stereoisomerically pure form(e.g., geometrically pure, enantiomerically pure or diastereomericallypure) and enantiomeric and stereoisomeric mixtures. Enantiomeric andstereoisomeric mixtures can be resolved into their component enantiomersor stereoisomers using separation techniques or chiral synthesistechniques well known to the skilled artisan. The compounds may alsoexist in several tautomeric forms including the enol form, the keto formand mixtures thereof. Accordingly, the chemical structures depictedherein encompass all possible tautomeric forms of the illustratedcompounds. Similarly, the compounds described also include allisotopically labeled versions of the compounds where one or more atomshave an atomic mass different from the atomic mass conventionally foundin nature. Examples of isotopes that may be incorporated into thecompounds of the disclosure include, but are not limited to, ²H, ³H,¹³C, ¹⁴C, ¹⁵N, ¹⁸O, ¹⁷O, etc. Compounds may exist in unsolvated forms aswell as solvated forms, including hydrated forms and as N-oxides. Ingeneral, compounds may be hydrated, solvated or N-oxides. Certaincompounds may exist in multiple crystalline or amorphous forms. Ingeneral, all physical forms are equivalent for the uses contemplatedherein and are intended to be within the scope of the presentdisclosure. Further, it should be understood, when partial structures ofthe compounds are illustrated, that brackets indicate the point ofattachment of the partial structure to the rest of the molecule.

Biocatalytic Transamination Processes

The biocatalytic transamination processes described herein are based onengineered transaminase polypeptides that were prepared for theirability to carry out the transamination of the ketoamide substrate,4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-oneto the chiral amine product,(2R)-4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-amine,in the presence of an amino group donor. This chiral amine product,known as sitagliptin, is the active ingredient in JANUVIA® used for thetreatment of Type 2 diabetes. The corresponding naturally occurringtransaminase or the transaminase of SEQ ID NO:2 does not measurablyexhibit activity on the sitagliptin ketoamide substrate. The presentdisclosure shows that these engineered transaminases are also capable ofcarrying out transamination of other keto substrates to produce chiralamines in enantiomeric excess. The chiral amine products have thegeneral structure

wherein R¹ is optionally substituted aryl or heteroaryl and R² is anoptionally substituted alkyl, or an acyl, acyloxy, or alkoxy groupsattached to the chiral carbon by a substituted or unsubstitutedalkylene. The corresponding prochiral ketone substrates are contactedwith a transaminase under suitable reaction conditions to produce theamine product in enantiomeric excess. The resulting amine compounds canbe used in the synthesis of various drug products and isomers thereof,as further described below.

In some embodiments, the disclosure provides for a process for preparingan amine product of structural formula (I):

having the indicated stereochemical configuration at the stereogeniccenter marked with an *; in an enantiomeric excess over the oppositeenantiomer, wherein

R¹ is optionally substituted aryl or heteroaryl; and

R² is an optionally substituted C₁-C₆ alkyl, —R³C(O)R⁴, or —R³OC(O)R⁵

wherein R³ is an optionally substituted C₁-C₄ alkyl, and R⁴ is H, anoptionally substituted C₁-C₄ alkyl, NR⁶R⁷, or OR⁸, where R⁵, R⁶, R⁷, andR⁸ are independently H or C₁-C₄ alkyl, and which process comprisescontacting a ketone substrate of structural formula (II):

with a transaminase polypeptide in presence of an amino donor underreaction conditions suitable for converting the ketone substrate to theamine product, wherein the transaminase polypeptide is an engineeredtransaminase described herein. In some embodiments, the transaminasepolypeptide has at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identityto SEQ ID NO:4 and is capable of converting the ketone substrate offormula (II) to the amine product of formula (I) at a rate that isimproved as compared to the transaminase of SEQ ID NO:2.

In some embodiments, the transaminase polypeptide has at least 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore amino acid sequence identity to SEQ ID NO: 58, 72, 74, 80, 86, 96,98, 100, or 102, and is capable of converting the ketone substrate tothe amine product at a rate that is improved as compared to thetransaminase of SEQ ID NO:2. Specific embodiments of the engineeredtransaminases for use in the processes are further described below.

In some embodiments, the engineered transaminase has at least 5%, 10%,20%, 30%, 40%, 50% or more activity of SEQ ID NO:74.

In some embodiments, the amine product is produced in at least 70%, 80%,85%, 90%, 95%, 96%, 97%, 96%, or 99% or more enantiomeric excess. Insome embodiments of the processes described above, the amine product isproduced in at least 99% enantiomeric excess.

As mentioned above, the transaminase polypeptides useful in theprocesses of the present disclosure can be characterized in terms of theability to convert the sitagliptin ketoamide substrate to thesitagliptin. Accordingly, in any of the embodiments of the processesdisclosed herein, the process can be carried out wherein thetransaminase polypeptide is capable of converting the sitagliptinketoamide substrate to the sitagliptin at a rate that is improved ascompared to the transaminase of SEQ ID NO:2, and has at least 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore amino acid sequence identity to SEQ ID NO:4.

In some embodiments of the process above, R¹ is an optionallysubstituted phenyl. In some embodiments R¹ is an optionally substitutedpyridinyl. In some embodiments, R¹ is a substituted aryl or heteroaryl.In some embodiments, the substitutions on the aryl or heteroaryl areselected from C₁-C₄ alkyl, —OR′, —SR′, —NR′R′, —NO₂, —NO, —CN, —CF₃,halogen (e.g., —F, —Cl, —Br and —I), —C(O)R′, —C(O)OR′, —C(O)NR′,—S(O)₂R′, —S(O)₂NR′R″, where each R′ and R″ are independently selectedfrom the group consisting of hydrogen and (C₁-C₄) alkyl. In someembodiments, the (C₁-C₄) alkyl is a halo substituted alkyl.

In some embodiments, R² is a C₁-C₄ alkyl or halo substituted C₁-C₄alkyl, particularly methyl, halo substituted methyl, propyl or halosubstituted propyl. In some embodiments R² is CF₂H or CF₃.

In some embodiments of R², the substitution on the C₁-C₆ alkyl and R³groups are selected from halogen, OH, NR⁵R⁶, or OR⁸, where R⁵, R⁶ and R⁸are defined above. In some embodiments, the compounds of formula (I)produced in the process described above are substantially chirally purecompounds. In certain embodiments, the compounds of formula (I) producedin the process described above are chirally pure.

In some embodiments of the process, the amine product of formula (I) is:

wherein R⁹ is H, Cl, Br, F, CH₃, CF₃, NH₂, NO₂, CN, SCN, OCF₃, or OCH₃,and R² is an optionally substituted C₁-C₆ alkyl, and the ketonesubstrate of formula (II) is:

In some embodiments of the process, the amine product of formula (I) is:

and the ketone substrate of formula (II) is:

In some embodiments of the process, the amine product of formula (I) is:

and the ketone substrate of formula (II) is:

In some embodiments of the process, the amine product of formula (I) is:

and the ketone substrate of formula (II) is:

In some embodiments of the process, the amine product of formula (I) is:

and the ketone substrate of formula (II) is:

In some embodiments of the process, the amine product of formula (I) is:

and the ketone substrate of formula (II) is:

In some embodiments of the process, the amine product of formula (I) is:

and the ketone substrate of formula (II) is:

In some embodiments of the process, the amine product of formula (I) is:

wherein R⁹ as defined above, and the ketone substrate of formula (II) is

In some embodiments, the amine product in the foregoing process isproduced in at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 96%, or 99% ormore enantiomeric excess. In some embodiments, the compounds produced inthe foregoing process are substantially chirally pure compounds. In someembodiments, the compounds produced in the foregoing process arechirally pure compounds.

In some embodiments of the process, the amine product of formula (I) is:

wherein R⁹ as defined above, and the ketone substrate of formula (II) is

In some embodiments of the process, the amine product of formula (I) is:

wherein R⁹ is H, Cl, Br, F, CH₃, CF₃, NH₂, NO₂, CN, SCN, OCF₃, or OCH₃,and the ketone substrate of formula (II) is:

In some embodiments R⁹ is H, Br, CH₃, or CF₃. In some embodiments R⁹ isin the para position on the phenyl ring. In some embodiments, the amineproduct in the foregoing process is produced in at least 70%, 80%, 85%,90%, 95%, 96%, 97%, 96%, or 99% or more enantiomeric excess. In someembodiments, the compounds produced in the foregoing process aresubstantially chirally pure compounds. In some embodiments, thecompounds produced in the foregoing process are chirally pure compounds.

In some embodiments of the process, the amine product of formula (I) is(S)-1-(4-bromophenyl)-2,2,2-trifluoroethanamine:

and the ketone substrate of formula (II) is1-(4-bromophenyl)-2,2,2-trifluoroethanone:

In some embodiments of the process, the(S)-1-(4-bromophenyl)-2,2,2-trifluoroethanamine is produced in at least70%, 80%, 85%, 90%, 95%, 96%, 97%, 96%, or 99% or more enantiomericexcess. In some embodiments, the(S)-1-(4-bromophenyl)-2,2,2-trifluoroethanamine produced issubstantially chirally pure compounds. In certain embodiments, the(S)-1-(4-bromophenyl)-2,2,2-trifluoroethanamine produced in the processis chirally pure.

In some embodiments of the process, the amine product of formula (I) is(S)-2,2,2-trifluoro-1-p-tolylethanamine:

and the ketone substrate of formula (II) is2,2,2-trifluoro-1-p-tolylethanone

In some embodiments, the (S)-2,2,2-trifluoro-1-p-tolylethanamine isproduced in at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 96%, or 99% ormore enantiomeric excess. In some embodiments, the(S)-2,2,2-trifluoro-1-p-tolylethanamine produced is substantiallychirally pure. In certain embodiments, the(S)-2,2,2-trifluoro-1-p-tolylethanamine produced in the process ischirally pure.

In some embodiments of the process, the product amine of formula (I) is(S)-2,2,2-trifluoro-1-(4-(trifluoromethyl)phenyl)ethanamine:

and the ketone substrate of formula (II) is2,2,2-trifluoro-1-(4-(trifluoromethyl)phenyl)ethanone:

In some embodiments, the(S)-2,2,2-trifluoro-1-(4-(trifluoromethyl)phenyl)ethanamine is producedin at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 96%, or 99% or moreenantiomeric excess. In some embodiments, the(S)-2,2,2-trifluoro-1-(4-(trifluoromethyl)phenyl)ethanamine produced issubstantially chirally pure. In certain embodiments, the(S)-2,2,2-trifluoro-1-(4-(trifluoromethyl)phenyl)ethanamine produced inthe process is chirally pure.

In some embodiments of the process, the product amine of formula (I) is:

and the ketone substrate of formula (II) is:

wherein R⁷ is an optionally substituted C₁-C₄ alkyl and R¹⁰ is hydrogen,halogen, amino or substituted amino, C₁-C₄ alkyl, halo substituted C₁-C₄alkyl, nitro, cyano, thiocyano, or alkoxy. In some embodiments, R¹⁰ isR⁹ described above. In some embodiments, R¹⁰ is H or F. In someembodiments, R⁷ is C₁-C₄ alkyl.

In some embodiments, the amine product in the foregoing process isproduced in at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 96%, or 99% ormore enantiomeric excess. In some embodiments, the compounds produced inthe foregoing process are substantially chirally pure compounds. In someembodiments, the compounds produced in the foregoing process arechirally pure compounds.

In some embodiments of the process, the amine product of formula (I) is(R)-ethyl-3-amino-3-(pyridin-2-yl)propanoate:

and the ketone substrate of formula (II) is ethyl3-oxo-3-(pyrindin-2-yl)propanoate:

In some embodiments, the (R)-ethyl-3-amino-3-(pyridin-2-yl)propanoate isproduced in at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 96%, or 99% ormore enantiomeric excess. In some embodiments, the(R)-ethyl-3-amino-3-(pyridin-2-yl)propanoate produced is substantiallychirally pure. In certain embodiments, the(R)-ethyl-3-amino-3-(pyridin-2-yl)propanoate produced in the process ischirally pure.

In some embodiments of the process, the amine product of formula (I) is:

wherein R¹¹ is halogen, OH, —C(O)R⁴, —OC(O)R⁵, or NR⁶R⁷, where R⁴, R⁵,R⁶, R⁷, and R¹⁰ are defined above, and the ketone substrate of formula(II) is:

In some embodiments, the amine product in the foregoing process isproduced in at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 96%, or 99% ormore enantiomeric excess. In some embodiments, the compounds produced inthe foregoing process are substantially chirally pure compounds. In someembodiments, the compounds produced in the foregoing process arechirally pure compounds.

In some embodiments the amine product of formula (I) is(S)-4-chloro-1-(2-fluorophenyl)butan-1-amine:

and the ketone substrate of formula (II) is4-chloro-1-(2-fluorophenyl)butan-1-one:

In some embodiments, the (S)-4-chloro-1-(2-fluorophenyl)butan-1-amine isproduced in at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 96%, or 99% ormore enantiomeric excess. In some embodiments, the(S)-4-chloro-1-(2-fluorophenyl)butan-1-amine produced is substantiallychirally pure. In certain embodiments, the(S)-4-chloro-1-(2-fluorophenyl)butan-1-amine produced in the process ischirally pure.

In some embodiments, the process using the transaminase biocatalysts canbe used for preparing the compound of formula (III):

having the indicated stereochemical configuration at the stereogeniccenter marked with an *, in an enantiomeric excess over the oppositeenantiomer, wherein R¹⁰ is as defined above. The process for preparingthe amine product of compound (III) can comprise:

(a) contacting a ketone substrate of formula

wherein R¹⁰ and R¹¹ are as defined above, with a transaminasepolypeptide described herein in presence of an amino donor underreaction conditions suitable for converting the ketone substrate to anamine product of formula:

and

(b) cyclizing the amine product under suitable conditions to form thecompound of formula (III).

In some embodiments, the cyclized amine product for formula (III) in theforegoing process is produced in at least 70%, 80%, 85%, 90%, 95%, 96%,97%, 96%, or 99% or more enantiomeric excess. In some embodiments, thecompounds produced in the foregoing process are substantially chirallypure compounds. In some embodiments, the compounds produced in theforegoing process are chirally pure compounds.

In some embodiments the ketone substrate is4-chloro-1-(2-fluorophenyl)butan-1-one:

and the amine product is (S)-4-chloro-1-(2-fluorophenyl)butan-1-amine:

thereby forming (R)-2-(2-fluorophenyl)pyrrolidine:

in enantiomeric excess.

In some embodiments, the (R)-2-(2-fluorophenyl)pyrrolidine is producedin at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 96%, or 99% or moreenantiomeric excess. In some embodiments, the(R)-2-(2-fluorophenyl)pyrrolidine produced is substantially chirallypure. In certain embodiments, the (R)-2-(2-fluorophenyl)pyrrolidineproduced in the process is chirally pure.

In some embodiments of the process, the amine product of formula (I) is:

wherein R⁹ as defined above, and the ketone substrate of formula (II) is

In some embodiments, the amine product in the foregoing process isproduced in at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 96%, or 99% ormore enantiomeric excess. In some embodiments, the compounds produced inthe foregoing process are substantially chirally pure compounds. In someembodiments, the compounds produced in the foregoing process arechirally pure compounds.

In some embodiments of the process, the amine product of formula (I) is:

wherein R⁹ as defined above, and the ketone substrate of formula (II) is

In some embodiments, the amine product in the foregoing process isproduced in at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 96%, or 99% ormore enantiomeric excess. In some embodiments, the compounds produced inthe foregoing process are substantially chirally pure compounds. In someembodiments, the compounds produced in the foregoing process arechirally pure compounds.

In some embodiments of the process, the amine product of formula (I) is:

wherein R⁹ as defined above, and the ketone substrate of formula (II) is

In some embodiments, the amine product in the foregoing process isproduced in at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 96%, or 99% ormore enantiomeric excess. In some embodiments, the compounds produced inthe foregoing process are substantially chirally pure compounds. In someembodiments, the compounds produced in the foregoing process arechirally pure compounds.

In some embodiments of the process, the amine product of formula (I) is:

wherein R⁹ as defined above, and the ketone substrate of formula (II) is

In some embodiments, the amine product in the foregoing process isproduced in at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 96%, or 99% ormore enantiomeric excess. In some embodiments, the compounds produced inthe foregoing process are substantially chirally pure compounds. In someembodiments, the compounds produced in the foregoing process arechirally pure compounds.

In some embodiments, the amine product in the foregoing process isproduced in at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 96%, or 99% ormore enantiomeric excess. In some embodiments, the compounds produced inthe foregoing process are substantially chirally pure compounds. In someembodiments, the compounds produced in the foregoing process arechirally pure compounds.

In some embodiments of the process, the amine product of formula (I) is:

wherein R⁹ is defined above, and the ketone substrate of formula (II)is:

In some embodiments of the process, the amine product of formula (I) is:

wherein R⁹ as defined above, and the ketone substrate of formula (II)is:

In some embodiments, the amine product in the foregoing process isproduced in at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 96%, or 99% ormore enantiomeric excess. In some embodiments, the compounds produced inthe foregoing process are substantially chirally pure compounds. In someembodiments, the compounds produced in the foregoing process arechirally pure compounds.

As noted above, the processes described above are carried out inreaction conditions suitable for converting the ketone substrate to thecorresponding chiral amine product in enantiomeric excess. In someembodiments, the reaction condition comprises a temperature of about 20°C. to about 65° C. In some embodiments the reaction condition comprisesa temperature of about 40° C. to about 65° C. In some embodiments thereaction condition comprises a temperature of about 50° C. to about 65°C. For instance, for the process using the following ketone substrate:

the reaction condition comprises a temperature of 40° C. to 65° C. Anexemplary temperature is 60° C.

In some embodiments the reaction condition under which the processesdescribed above are done comprises a pH of about 7.0 to about 11.0. Insome embodiments the reaction condition comprises a pH of about 7.0 toabout 9.0. In some embodiments, the reaction condition for the processis a pH of about 8.5. While the pH can be adjusted during the processusing any base and/or acid, in some embodiments, the pH can bemaintained by adding isopropylamine, which also provides a source ofamino donor to push the reaction equilibrium toward amine productformation.

Various organic solvents can be used in the process to facilitate theenzyme reaction and to place the substrate and/or product in solution.In some embodiments of the process, the organic solvent comprises apolar solvent, such as methanol or dimethylsulfoxide (DMSO). In someembodiments, the organic solvent is DMSO, which can be present fromabout 10% to about 40% volume/volume (v/v). In some embodiments, theorganic solvent is DMSO, which can be present from about 10% to about50% volume/volume (v/v). In some embodiments, the DMSO is present atabout 40% v/v.

As discussed above, the amino group donor used in the process can be achiral amine or an achiral amine. An achiral amino group donor has theadvantage of not being limited in its reaction to a specificstereoisomer, thus requiring less of the amino group donor. Varioussuitable amino group donors can be used, including, by way of exampleand not limitation, isopropylamine (also referred to as 2-aminopropane),L, D or DL alanine, phenylalanine, glutamate, glutamine, leucine (or anyother suitable α-amino acids), 3-aminobutyric acid (or any othersuitable β-amino acids), and methylbenzylamine. In some embodiments, theamino group donor is isopropylamine. In some embodiments, other aminogroup donors may be used, including, among others, α-phenethylamine(also termed 1-phenylethanamine), and its enantiomers(S)-1-phenylethanamine and (R)-1-phenylethanamine,2-amino-4-phenylbutane, glycine, L-glutamic acid, L-glutamate,monosodium glutamate, L-aspartic acid, L-lysine, L-ornithine, β-alanine,taurine, n-octylamine, cyclohexylamine, 1,4-butanediamine,1,6-hexanediamine, 6-aminohexanoic acid, 4-aminobutyric acid, tyramine,and benzyl amine, 2-aminobutane, 2-amino-1-butanol,1-amino-1-phenylethane, 1-amino-1-(2-methoxy-5-fluorophenyl)ethane,1-amino-1-phenylpropane, 1-amino-1-(4-hydroxyphenyl)propane,1-amino-1-(4-bromophenyl)propane, 1-amino-1-(4-nitrophenyl)propane,1-phenyl-2-aminopropane, 1-(3-trifluoromethylphenyl)-2-aminopropane,2-aminopropanol, 1-amino-1-phenylbutane, 1-phenyl-2-aminobutane,1-(2,5-dimethoxy-4-methylphenyl)-2-aminobutane, 1-phenyl-3-aminobutane,1-(4-hydroxyphenyl)-3-aminobutane, 1-amino-2-methylcyclopentane,1-amino-3-methylcyclopentane, 1-amino-2-methylcyclohexane,1-amino-1-(2-naphthyl)ethane, 3-methylcyclopentylamine,2-methylcyclopentylamine, 2-ethylcyclopentylamine,2-methylcyclohexylamine, 3-methylcyclohexylamine, 1-aminotetralin,2-aminotetralin, 2-amino-5-methoxytetralin, and 1-aminoindan, includingboth (R) and (S) single isomers where possible and including allpossible salts of the amines.

In some embodiments of the processes above, the step in the process canfurther comprise removal of the carbonyl by-product formed from theamino group donor when the amino group is transferred to the amino groupacceptor. Such removal in situ can reduce the rate of the reversereaction such that the forward reaction dominates and more substrate isthen converted to product.

Removal of the carbonyl by-product can be carried in a number of ways.Where the amino group donor is an amino acid, such as alanine, thecarbonyl by product, a keto acid, can be removed by reaction with aperoxide (see, e.g., US 2008/0213845, incorporated herein by reference).Peroxides which can be used include, among others, hydrogen peroxide;peroxyacids (peracids) such as peracetic acid (CH₃CO₃H),trifluoroperacetic acid and metachloroperoxybenzoic acid; organicperoxides such as t-butyl peroxide ((CH₃)₃COOH), or other selectiveoxidants such as tetrapropylammonium perruthenate, MnO₂, KMnO₄,ruthenium tetroxide and related compounds. Alternatively, pyruvateremoval can be achieved via its reduction to lactate by employinglactate dehydrogenase to shift equilibrium to the product amine (see,e.g., Koszelewski et al., 2008, Adv. Syn. Catal. 350: 2761-2766).Pyruvate removal can also be achieved via its decarboxylation to carbondioxide acetaldehyde by employing pyruvate decarboxylase (see e.g.,Höhne et al., 2008, ChemBioChem 9: 363-365).

In some embodiments, where the choice of the amino group donor resultsin a carbonyl by-product that has a vapor pressure higher than water(e.g., a low boiling co-product such as a volatile organic carbonylcompound), the carbonyl by-product can be removed by sparging thereaction solution with a non-reactive gas or by applying a vacuum tolower the reaction pressure and removing the carbonyl by-product presentin the gas phase. A non-reactive gas is any gas that does not react withthe reaction components. Various non-reactive gases include nitrogen andnoble gases (e.g., inert gases). In some embodiments, the non-reactivegas is nitrogen gas.

In some embodiments, the amino acid donor used in the process isisopropylamine, which forms the carbonyl by-product acetone upontransfer of the amino group to the amino group acceptor. The acetone canbe removed by sparging with nitrogen gas or applying a vacuum to thereaction solution and removing the acetone from the gas phase by anacetone trap, such as a condenser or other cold trap. Alternatively, theacetone can be removed by reduction to isopropanol using aketoreductase.

In some embodiments of the processes above where the carbonyl by-productis removed, the corresponding amino group donor can be added during thetransamination reaction to replenish the amino group donor and/ormaintain the pH of the reaction. Replenishing the amino group donor alsoshifts the equilibrium towards product formation, thereby increasing theconversion of substrate to product. Thus, in some embodiments whereinthe amino group donor is isopropylamine and the acetone product isremoved in situ, isopropylamine can be added to the solution toreplenish the amino group donor lost during the acetone removal and tomaintain the pH of the reaction (e.g., at about 8.5). Alternatively, inembodiments where an amino acid is used as amino group donor, the ketoacid carbonyl by-product can be recycled back to the amino acid byreaction with ammonia and NADH using an appropriate amino aciddehydrogenase enzyme, thereby replenishing the amino group donor.

The ketone substrate can be present in appropriate amounts, depending onfactors, among others, such as the nature of solvent, the stability ofthe transaminase to reaction temperature, the amount and activity of theenzyme. In some embodiments, the substrate is present at 5 to 50 g/L. Insome embodiments, the substrate is present at 5-25 g/L.

In some embodiments, the process described above comprises contactingthe keto substrate at about 10 to 50 g/L with about 1 to 20 g/L of atransaminase described herein under reaction conditions of pH 7.5 to 9.0and a temperature of 40 to 60° C. in presence of isopropylamine of fromabout 1 M to about 2 M, wherein at least 80%, 85%, 90%, 92%, 94%, 96%,or 98% or more of the substrate is converted to product in 24 hrs. Insome embodiments, the transaminase polypeptide capable of carrying outthe foregoing reaction comprises an amino acid sequence corresponding toSEQ ID NO: 58, 72, 74, 80, 86, 96, 98, 100, 102, 110 or 166.

In some embodiments, the processes above can further comprise the stepof isolating the amine products, such as the amine products ofstructural formulas (I), (III), or (V), from the reaction mixture.

Also provided herein are compositions of the transaminases andsubstrates/products. In some embodiments, the compositions can comprisethe amine products of structural formulas (I), (III), or (V) and atransaminase of the disclosure. Any one or more of the engineeredtransaminases can be part of the composition.

In some embodiments, the compositions can further comprise an aminogroup donor. In some embodiments of the compositions, the amino groupdonor can comprise isopropylamine, alanine, 3-aminobutyric acid, ormethylbenzylamine. In some embodiments of the compositions, the aminogroup donor is an isopropylamine.

The processes of the disclosure are useful for producing variousintermediates for the synthesis of isomers and derivatives ofpharmaceutical molecules. For example, the processes of the disclosurethat produce compounds of formula (I), can be used in the synthesis ofmolecules such as Odanacatib or related derivatives, an investigationalselective cathepsin K inhibitor for the prevention of bone turnover incancer patients. Odanacatib has the following structure:

The processes that produce compounds of formula (I) can be used to makeintermediates for synthesis of certain thiadaizoles such as the moleculebelow. The thiadaizoles are CXC- and CC-chemokine receptor ligands,purported to have anti-inflammatory and anti-tumor properties (WO2005/066147).

The processes of the disclosure that produce compounds of formula (V)can be used to synthesize compounds such as:

WO 2008/128647 discloses similar quinoline-carboxamide derivatives asP2Y12 antagonists which are potentially useful in the treatment ofcardiovascular disorders.

Transaminase Polypeptides and Polynucleotides

As noted above, the transaminase polypeptides used in theabove-described methods were initially identified based on their abilityto convert the ketoamide substrate4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-one(“the sitagliptin ketoamide substrate”) to the product(2R)-4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-aminefor the synthesis of sitagliptin. The transaminase polypeptide of SEQ IDNO; 2 does not efficiently carry out the transamination reaction. Thesetransaminases polypeptides can be described in relation to theiractivity toward the sitagliptin ketoamide substrate, or to thesubstrates described herein.

Transaminases, including those described herein, typically contain acoenzyme, pyridoxal phosphate (PLP), which participates in thetransamination reaction. PLP can be provided by the host cell in whichthe polypeptide is synthesized, or provided by adding PLP to a solutionof the polypeptide. While the transaminase is described with respect tothe amino acid sequence, it will be understood by those skilled in theart that the active polypeptide contains PLP or a suitable analog as acoenzyme.

As noted above, in some embodiments, the transaminase polypeptide has atleast 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more amino acid sequence identity to SEQ ID NO:4 and iscapable of converting the ketone substrate to the amine product at arate that is improved as compared to the transaminase of SEQ ID NO:2.

In some embodiments, the transaminase polypeptide has at least 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore amino acid sequence identity to SEQ ID NO: 58, 72, 74, 80, 86, 96,98, 100, or 102, and is capable of converting the ketone substrate tothe amine product at a rate that is improved as compared to thetransaminase of SEQ ID NO:2.

In some embodiments, the engineered transaminase polypeptides comprisean amino acid sequence that has one or more residue differences ascompared to a transaminase reference sequence. The residue differencescan be non-conservative substitutions, conservative substitutions, or acombination of non-conservative and conservative substitutions. Withrespect to the residue differences and the descriptions of residuepositions, the transaminases provided herein can be described inreference to the amino acid sequence of the naturally occurringtransaminase of Arthrobacter sp KNK168, or the transaminase of SEQ IDNO:2, or another engineered transaminase, such as the polypeptide of SEQID NO:4. For the descriptions herein, the amino acid residue position inthe reference sequence is determined in the transaminase beginning fromthe initiating methionine (M) residue (i.e., M represents residueposition 1), although it will be understood by the skilled artisan thatthis initiating methionine residue may be removed by biologicalprocessing machinery, such as in a host cell or in vitro translationsystem, to generate a mature protein lacking the initiating methionineresidue.

The polypeptide sequence position at which a particular amino acid oramino acid change (“residue difference”) is present is sometimesdescribed herein as “Xn”, or “position n”, where n refers to the residueposition with respect to the reference sequence.

A specific substitution mutation, which is a replacement of the specificresidue in a reference sequence with a different specified residue maybe denoted by the conventional notation “X(number)Y”, where X is thesingle letter identifier of the residue in the reference sequence,“number” is the residue position in the reference sequence, and Y is thesingle letter identifier of the residue substitution in the engineeredsequence.

In some embodiments, the residue differences can occur at one or more ofthe following residue positions: X4; X5; X8; X18; X25; X26; X27; X28;X30; X41; X42; X48; X49; X50; X54; X55; X60; X61; X62; X65; X69; X81;X94; X96; X102; X117; X120; X122; X124; X126; X136; X137; X138; X146;X148; X150; X152; X155; X156; X160; X163; X164; X169; X174; X178; X195;X199; X204; X208; X209; X211; X215; X217; X223; X225; X230; X252; X269;X273; X282, X284; X292; X297; X302; X306; X321; and X329. In someembodiments, the residue differences or combinations thereof, areassociated with the improved enzyme properties. In some embodiments, thetransaminase polypeptides can have additionally 1-2, 1-3, 1-4, 1-5, 1-6,1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22,1-24, 1-26, 1-30, 1-35, 1-40, 1-45, 1-50, 1-55, or 1-60 residuedifferences at residue positions other than those specific positionsdenoted by “Xn” listed above. In some embodiments, the number ofdifferences can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16,18, 20, 22, 24, 26, 30, 35, 40, 45, 50, 55, or 60 residue differences atthe other amino acid residue positions. In some embodiments, the residuedifferences at other residue positions comprise substitutions withconservative amino acid residues.

In the embodiments herein, the residue differences as compared to SEQ IDNO:2 at residue positions affecting substrate binding on thetransaminase allows accommodation of various ketoamide substrates.Without being bound by theory, at least two regions, a first substratebinding region and a second substrate binding region, interact withdifferent structural elements of ketoamide substrates. The first bindingregion comprises residue positions X62, X136, X137, X195, X199, X208,X209, X223, X225, and X282, while the second binding region comprisesresidue positions X69, X122, and X284. Accordingly, the transaminasepolypeptides herein have one or more residue differences at residuepositions comprising X62, X69, X122, X136, X137, X195, X199, X208, X209,X223, X225, X282, and X284. In some embodiments, the transaminasepolypeptides herein have at least two or more, three or more, four ormore, five or more, or six or more residue differences at the specifiedresidue positions associated with substrate binding.

In some embodiments, the residue differences as compared to SEQ ID NO:2are at one or more of residue positions forming a first substratebinding region comprised of residue positions X62, X136, X137, X195,X199, X208, X209, X223, X225, and X282. Accordingly, in someembodiments, the engineered transaminase comprises an amino acidsequence that includes at least one residue difference as compared toSEQ ID NO:2 at residue positions X62, X136, X137, X195, X199, X208,X209, X223, X225, and X282.

In some embodiments, the residue differences as compared to SEQ ID NO:2are at one or more of residue positions forming a second substratebinding region comprised of residue positions X69, X122, and X284.Accordingly, in some embodiments, the engineered transaminase comprisesan amino acid sequence that includes at least one residue difference ascompared to SEQ ID NO:2 at residue positions X69, X122, and X284.

In some embodiments, the engineered transaminase comprises an amino acidsequence that includes residue differences at the first binding regionin combination with residue differences at the second binding region.Accordingly, in some embodiments, the engineered transaminase comprisesan amino acid sequence that includes one or more residue difference ascompared to SEQ ID NO:2 at residue positions X62, X136, X137, X195,X199, X208, X209, X223, X225, and X282 in combination with one or moreresidue difference as compared to SEQ ID NO:2 at residue positions X69,X122, and X284.

In some embodiments of the engineered transaminases of the disclosure,the amino acid residues at a residue position can be can be defined interms of the amino acid “features” (e.g., type or property of aminoacids) that can appear at that position. Thus, in some embodiments theamino acid residues at the positions specified above can be selectedfrom the following features: X4 is an aromatic residue; X5 is a basicresidue; X8 is a constrained residue; X18 is a cysteine (C) or analiphatic residue; X25 is a polar residue; X26 is an aromatic orconstrained residue; X27 is a polar residue; X28 is a constrainedresidue; X30 is a polar or non-polar residue; X41 is a constrained orpolar residue; X42 is a non-polar residue; X48 is a polar, acidic,aliphatic or non-polar residue; X49 is a polar residue; X50 is analiphatic residue; X54 is a constrained residue; X55 is an aliphaticresidue; X60 is an aromatic residue; X61 is an aromatic residue; X62 isan aromatic or polar residue; X65 is an aliphatic residue; X69 is acysteine (C) or non-polar, polar, or aliphatic residue; X81 is anon-polar residue; X94 is an aliphatic residue; X96 is an aliphaticresidue; X102 is an aliphatic or basic residue; X117 is a non-polarresidue; X120 is an aromatic residue; X122 is a constrained, non-polaror aliphatic residue; X124 is a polar or constrained residue; X126 is apolar residue; X136 is an aromatic residue; X137 is a polar or aliphaticresidue; X138 is a basic or constrained residue; X146 is a basicresidue; X148 is an aliphatic or aromatic residue; X150 is aromatic,constrained or polar residue; X152 is a cysteine (C), non-polar,aliphatic, or polar residue; X155 is a non-polar or polar residue; X156is a polar residue; X160 is an aliphatic residue; X163 is an aliphaticor constrained residue; X164 is an aliphatic or constrained residue;X169 is an aliphatic residue; X174 is an aliphatic residue; X178 is apolar residue; X195 is an aromatic or polar residue; X199 is analiphatic or aromatic residue; X204 is an aliphatic residue; X208 is acysteine (C) or a constrained, non-polar, aromatic, polar, or basicresidue; X209 is an aliphatic residue; X211 is an aliphatic residue;X215 is a cysteine (C); X217 is a polar residue; X223 is a constrainedresidue; X225 is an aromatic residue; X230 is an aliphatic residue; X252is an aromatic or aliphatic residue; X269 is a constrained residue; X273is an aromatic residue; X282 is a polar residue; X284 is a non-polarresidue; X292 is a polar residue; X297 is a polar residue; X302 is analiphatic residue; X306 is an aliphatic residue; X321 is a constrainedresidue, and X329 is a constrained or aromatic residue. In someembodiments, where the amino acid residue at the corresponding residueposition of the reference sequence are encompassed within the categoryof amino acids described for the specified position, a different aminoacid within that amino acid category can be used in light of theguidance provided herein.

In some embodiments, the amino acid residue at the residue positionsspecified above can be selected from the following features: X4 is Y, F,or W, particularly Y; X5 is K or R, particularly K; X8 is H or P,particularly P; X18 is C, A, V, or I, particularly C or I; X25 is N, Q,S, or T, particularly Q; X26 is F, W, H or P, particularly H; X27 is N,Q, S, or T, particularly T; X28 is P or H, particularly P; X30 is N, Q,S, T, G, M, A, V, L or I, particularly Q or M; X41 is P, H, N, Q, S, orT, particularly H or S; X42 is G, M, A, V, L or I, particularly G; X48is N, Q, S, T, D, E, G, M, A, V, L, or I, particularly Q, D, V, G, or A;X49 is N, Q, or T, particularly T; X50 is A, V, L or I, particularly L;X54 is P or H; X55 is A, V, or L, particularly V; X60 is F or W,particularly F; X61 is Y, F, or W, particularly Y; X62 is S, T, N, Q, Y,F, or W, particularly T, Y or F; X65 is A, L or I, particularly A; X69is C, G, M, A, L I, S, T, N or Q, particularly G, C, T, A, or S; X81 isG, M, A, V, L, I, particularly G; X94 is A, V, L or I, particularly I orL; X96 is A, V or L, particularly L; X102 is A, V, L, I, K or R,particularly L or K; X117 is G, M, A, V, L or I, particularly G; X120 isY, W, or F, particularly Y; X122 is G, M, A, V, I, L, P or H,particularly M, I, L, V, or H; X124 is T, N, Q, P, or H, particularly T,H or N; X 126 is N, Q, or T, particularly T; X136 is Y, F or W,particularly Y or F; X137 is S, T, N, Q, A, V, L or I, particularly T orI; X138 is K, P or H, particularly K or P; X146 is K or R, particularlyR; X148 is A, V, L I, W, or F, particularly A or F; X150 is F, W, H, P,S, T, N, or Q, particularly F, H, or S; X152 is C, G, M, A, L, I, S, T,N, or Q, particularly I, L, S or C; X155 is N, S, T, G, M, A, V, L or I,particularly M, V or T; X156 is N, Q, S, or T, particularly Q; X160 isA, V, L or I, particularly L; X163 is P, H, A, V, or L, particularly Hor V; X164 is A, V, L, I, P or H, particularly V or P; X169 is V, L orI, particularly L; X174 is A, V, L or I, particularly A; X178 is S, N,or Q, particularly S; X195 is F, Y, W, S, T, N or Q, particularly F orQ; X199 is A, L, I, Y, F, W, particularly W or I; X204 is A, V, L, or I,particularly A; X208 is H, C, G, K, N, Y, D or S; X209 is V, L or I,particularly L; X211 is A, V, or I, particularly I; X215 is C; X217 isS, T, N or Q, particularly N; X223 is H or P, particularly P; X225 is Wor Y, particularly Y; X230 is A, V, or L, particularly V; X252 is A, V,I, Y, F, or W, particularly F; X269 is H or P, particularly P; X273 isY, F or W, particularly Y; X282 is S, N or Q, particularly S; X284 is G,M, V, L or I, particularly G; X292 is T, N, or Q, particularly T; X297is S, T, N or Q, particularly S; X302 is A, L, or I, particularly A;X306 is A, L or I, particularly L; X321 is H or P, particularly P; andX329 is H, P, Y, F, or W, particularly H.

In some embodiments, the amino acid residue at the residue positionsspecified above can be selected from the following features: X4 is Y; X5is K; X8 is P; X18 is C or I; X25 is Q; X26 is H; X27 is T; X28 is P;X30 is Q or M; X41 is H or S; X42 is G; X48 is Q, D, V, G, or A; X49 isT; X50 is L; X54 is P or H; X55 is V; X60 is F; X61 is Y; X62 is T, Y orF; X65 is A; X69 is G, C, T, A, or S; X81 is G; X94 is I or L; X96 is L;X102 is L or K; X117 is G; X120 is Y; X122 is M, I, L, V, or H; X124 T,H or N; X 126 is T; X136 is Y or F; X137 is T or I; X138 is K or P; X146is R; X148 is A or F; X150 is F, H, or S; X152 is I, L, S or C; X155 isM, V or T; X156 is Q; X160 is L; X163 is H or V; X164 is V or P; X169 isL; X174 is A; X178 is S; X195 is F or Q; X199 is W or I; X204 is A, V,L, or I, particularly A; X208 is H, C, G, K, N, Y, D or S; X209 is L;X211 is I; X215 is C; X217 is N; X223 is P; X225 is Y; X230 is V; X252is F; X269 is P; X273 is Y; X282 is S; X284 is G; X292 is T; X297 is S;X302 is A; X306 is L; X321 is P; and X329 is H.

In some embodiments, the amino acid residue at the residue positionsspecified above can be selected from the following features: X8 is P;X60 is F; X61 is Y; X62 is T, Y or F; X65 is A; X69 is G, C, T, A, or S;X81 is G; X94 is I or L; X96 is L; X122 is M, I, L, V, or H; X124 T, Hor N; X136 is Y or F; X169 is L; X178 is S; X199 is W or I; X209 is L;X215 is C; X217 is N; X223 is P; X269 is P; X273 is Y; X282 is S; X284is G; X297 is S; X321 is P and X329 is H.

In some embodiments, the engineered transaminase polypeptide comprisesan amino acid sequence that includes one or more of the followingfeatures: residue corresponding to X69 is cysteine (C) or a non-polar,polar, or aliphatic residue; residue corresponding to X122 is aconstrained, non-polar or aliphatic residue; residue corresponding toX223 is a constrained residue; and residue corresponding to X284 is anon-polar residue.

In some embodiments, the engineered transaminase polypeptide comprisesan amino acid sequence that includes at least the following features:(1) residue corresponding to X69 is C or a non-polar, aliphatic or polarresidue, and/or residue corresponding to X284 is a non-polar residue;(2) residue corresponding to X122 is a constrained, non-polar oraliphatic residue; and (3) residue corresponding to X223 is aconstrained residue.

In some embodiments, the engineered transaminase polypeptide comprisesan amino acid sequence that includes at least the following features:X69 is C or a non-polar, aliphatic or polar residue; X122 is aconstrained, non-polar or aliphatic residue; and X223 is a constrainedresidue.

In some embodiments, the engineered transaminase polypeptide comprisesan amino acid sequence that includes at least the following features:X69 is C, G, M, A, L, I, S, T, N or Q, particularly G, C, T, A, or S;X122 is G, M, A, V, L, I, P or H, particularly M, I, V, L, or H; andX223 is H or P, particularly P.

In some embodiments, the engineered transaminase polypeptide comprisesan amino acid sequence that includes at least the following features:X122 is a constrained, non-polar or aliphatic residue; X223 is aconstrained residue; and X284 is a non-polar residue.

In some embodiments, the engineered transaminase polypeptide comprisesan amino acid sequence that includes at least the following features:X122 is G, M, A, V, L, I, P or H, particularly M, I, V, L or H; X223 isH or P particularly P; and X284 is G, M, V, L or I, particularly G.

In some embodiments, the engineered transaminase polypeptide comprisesan amino acid sequence that includes at least the following features:X69 is C or a non-polar, polar or aliphatic residue; X122 is aconstrained, non-polar or aliphatic residue; X223 is a constrainedresidue; and X284 is a non-polar residue.

In some embodiments, the engineered transaminase polypeptide comprisesan amino acid sequence that includes at least the following features:X69 is C, G, M, A, L, I, S, T, N or Q, particularly G, C, T, A, or S;X122 is G, M, A, V, L, I, P or H, particularly M, I, V, L, or H; X223 isH or P, particularly P; and X284 is G, M, A, V, L or I, particularly G.

In some embodiments, the engineered transaminase polypeptide comprisesan amino acid sequence that includes at least the following features:X69 is C or T; X122 is M or I; X223 is P; and X284 is G.

In some embodiments, the engineered transaminase polypeptides with oneor more of the specified features or combinations of features at residuepositions X69, X122, X223, and X284, can additionally have one or moreresidue differences as compared to SEQ ID NO:2 at the following residuepositions: X4; X5; X8; X18; X25; X26; X27; X28; X30; X41; X42; X48; X49;X50; X54; X55; X60; X61; X62; X65; X81; X94; X96; X102; X117; X120;X124; X126; X136; X137; X138; X146; X148; X150; X152; X155; X156; X160;X163; X164; X169; X174; X178; X195; X199; X204; X208; X209; X211; X215;X217; X225; X230; X252; X269; X273; X282; X292; X297; X306; X321; andX329. In addition to residue positions X69, X122, X223, and X284, theseother residue positions are associated with effects on variousproperties of the transaminase polypeptide, and thus can have residuedifferences as compared to SEQ ID NO:2 to effect desirable changes inenzyme properties.

As noted above, residue positions X62, X136, X137, X195, X199, X208,X209, X225, and X282 along with residue positions X69, X122, X223, andX284, are associated with binding of the substrate to the enzyme, andthus the transaminase polypeptide can have residue differences at theserecited positions as compared to SEQ ID NO:2 to effect desirable changesin enzyme activity.

Residue positions X4, X5, X8, X26, X48, X60, X65, X81, X96, X102, X124,X160, X163, X169, X174, X178, X211, X217, X225, X230, X252, X269, X273,X292, X297, X306, X321, X329 are also associated with additionalincreases in enzyme activity, and thus the transaminase polypeptide canhave residue differences at these recited positions as compared to SEQID NO:2 to effect additional desirable changes in enzyme activity, forexample increase in efficiency of conversion at high substrate loadingconditions.

Residue positions X18, X25, X27, X28, X30, X41, X42, X49, X50, X54, X55,X117, X120, X126, X138, X146, X148, X150, X152, X155, X156, X164, X204,X302 are associated also with increases in thermostability and/orsolvent stability, such as DMSO, and thus the transaminase polypeptidecan have residue differences at these recited positions as compared toSEQ ID NO:2 to effect desirable changes in thermostability and/orsolvent stability.

Residue positions X61, X94, X215 are associated also with the ability tocarry out the reaction at high concentrations of amino donorisopropylamine, and thus the transaminase polypeptide can have residuedifferences at these recited positions as compared to SEQ ID NO:2 toeffect increase in efficiency of conversion at high (e.g., 1-2 M)concentrations of isopropylamine.

It is to be understood that the residue differences from SEQ ID NO:2 atresidue positions associated with the various properties of the enzymescan be used in various combinations to form transaminase polypeptideshaving desirable enzymatic characteristics, for example combination ofincreases in enzyme activity, solvent and temperate stability, andutilization of amino donor. Exemplary combinations are described herein.

In some embodiments, the amino acid residues for the specified residuepositions can be selected according to the descriptions above. Forexample, the amino acid residues can be selected based on the followingfeatures: X4 is an aromatic residue; X5 is a basic residue; X8 is aconstrained residue; X18 is a cysteine (C) or an aliphatic residue; X25is a polar residue; X26 is an aromatic or constrained residue; X27 is apolar residue; X28 is a constrained residue; X30 is polar or non-polarresidue; X41 is a constrained or polar residue; X42 is non-polarresidue; X48 is a polar, acidic, aliphatic or non-polar residue; X49 isa polar residue; X50 is an aliphatic residue; X54 is a constrainedresidue; X55 is an aliphatic residue; X60 is an aromatic residue; X61 isan aromatic residue; X62 is an aromatic or polar residue; X65 is analiphatic residue; X81 is a non-polar residue; X94 is an aliphaticresidue; X96 is an aliphatic residue; X102 is an aliphatic or basicresidue; X117 is a non-polar residue; X120 is an aromatic residue; X124is a polar or constrained residue; X126 is a polar residue; X136 is anaromatic residue; X137 is a polar or aliphatic residue; X138 is a basicor constrained residue; X146 is a basic residue; X148 is an aliphatic oraromatic residue; X150 is aromatic, constrained or polar residue; X152is C, non-polar, aliphatic, or polar residue; X155 is non-polar or polarresidue; X156 is a polar residue; X160 is an aliphatic residue; X163 isan aliphatic or constrained residue; X164 is an aliphatic or constrainedresidue; X169 is an aliphatic residue; X174 is an aliphatic residue;X178 is a polar residue; X195 is an aromatic or polar residue; X199 isan aliphatic or aromatic residue; X204 is an aliphatic residue; X208 iscysteine (C) or a constrained, non-polar, aromatic, polar, or basicresidue; X209 is an aliphatic residue; X211 is an aliphatic residue;X215 is C; X217 is a polar residue; X225 is an aromatic residue; X230 isan aliphatic residue; X252 is an aromatic or aliphatic residue; X269 isa constrained residue; X273 is an aromatic residue; X282 is a polarresidue; X292 is a polar residue; X297 is a polar residue; X302 is analiphatic residue; X306 is an aliphatic residue; X321 is a constrainedresidue; and X329 is a constrained or aromatic residue. Specific aminoacid residues that can be used at these residue positions are describedabove.

In some embodiments, the engineered transaminase having the features atone or more residue positions X69, X122, X223, and X284 as describedabove, can have additionally one or more of the following features: X26is an aromatic or constrained residue; X61 is an aromatic residue; X62is an aromatic or polar residue; X65 is an aliphatic residue; X94 is analiphatic residue; X136 is an aromatic residue; X137 is a polar oraliphatic residue; X199 is an aliphatic or aromatic residue; X209 is analiphatic residue; X215 is C; and X282 is a polar residue.

In some embodiments, in addition to the preceding features, thetransaminase amino acid sequence can include additionally one or more ofthe following features: X8 is a constrained residue; X60 is an aromaticresidue; X81 is a non-polar or small residue; X96 is an aliphaticresidue; X124 is a polar or constrained residue; X169 is an aliphaticresidue; X217 is a polar residue; X269 is a constrained residue; X273 isan aromatic residue; X297 is a polar residue; and X321 is a constrainedresidue.

In some embodiments, in addition to the preceding features, thetransaminase amino acid sequence can include additionally one or more ofthe following features: X4 is an aromatic residue; X48 is a polar,acidic, aliphatic or non-polar residue; X102 is an aliphatic or basicresidue; X150 is aromatic, constrained or polar residue; X152 is C or anon-polar, aliphatic or polar residue; X160 is an aliphatic residue;X163 is an aliphatic or constrained residue; X174 is an aliphaticresidue; X178 is a polar residue; X195 is an aromatic or polar residue;X208 is C or a constrained, non-polar, aromatic, polar, or basicresidue; X211 is an aliphatic residue; X225 is an aromatic residue; X230is an aliphatic residue; X252 is an aromatic or aliphatic residue; X292is a polar residue; X306 is an aliphatic residue; and X329 is aconstrained or aromatic residue.

In some embodiments, the engineered transaminase having the features atone or more or combinations of features at residue positions X69, X122,X223, and X284 as described above, includes at least the followingadditional features: X26 is an aromatic or constrained residue, and/orX62 is an aromatic or polar residue; X65 is an aliphatic residue; X136is an aromatic residue; X199 is an aliphatic or aromatic residue; andX209 is an aliphatic residue.

In some embodiments, the engineered transaminase having the features atone or more residue positions X69, X122, X223, and X284 as describedabove, includes at least the following additional features: X61 is anaromatic residue; X62 is an aromatic or polar residue; X65 is analiphatic residue; X94 is an aliphatic residue; X136 is an aromaticresidue; X199 is an aliphatic or aromatic residue; X209 is an aliphaticresidue; X215 is C, and X282 is a polar residue.

In some embodiments, the engineered transaminase having the features atone or more residue positions X69, X122, X223, and X284 as describedabove, includes at least the following additional features: X8 is aconstrained residue; X61 is an aromatic residue; X62 is an aromatic orpolar residue; X65 is an aliphatic residue; X81 is a non-polar or smallresidue; X94 is an aliphatic residue; X136 is an aromatic residue; X199is an aliphatic or aromatic residue; X209 is an aliphatic residue; X215is a C; X217 is a polar residue; X269 is a constrained residue; X282 isa polar residue; X297 is a polar residue; and X321 is a constrainedresidue.

In some embodiments, the engineered transaminase having the features atone or more residue positions X69, X122, X223, and X284 as describedabove, includes at least the following additional features: X8 is aconstrained residue; X60 is an aromatic residue; X61 is an aromaticresidue; X62 is an aromatic or polar residue; X65 is an aliphaticresidue; X81 is a non-polar residue; X94 is an aliphatic residue; X96 isan aliphatic residue; X124 is a polar or constrained residue; X136 is anaromatic residue; X169 is an aliphatic residue; X199 is an aliphatic oraromatic residue; X209 is an aliphatic residue; X215 is C; X217 is apolar residue; X269 is a constrained residue; X273 is an aromaticresidue. X282 is a polar residue; X297 is a polar residue; and X321 is aconstrained residue.

In some embodiments, the engineered transaminase having the features atone or more residue positions X69, X122, X223, and X284 as describedabove, includes at least the following additional features: X8 is aconstrained residue; X60 is an aromatic residue; X61 is an aromaticresidue; X62 is an aromatic or polar residue; X65 is an aliphaticresidue; X81 is a non-polar residue; X94 is an aliphatic residue; X96 isan aliphatic residue; X124 is a polar or constrained residue; X126 is apolar residue; X136 is an aromatic residue; X150 is an aromatic,constrained or polar residue; X152 is a cysteine (C), non-polar,aliphatic, or polar residue; X169 is an aliphatic residue; X199 is analiphatic or aromatic residue; X209 is an aliphatic residue; X215 is C;X217 is a polar residue; X269 is a constrained residue; X273 is anaromatic residue. X282 is a polar residue; X297 is a polar residue; andX321 is a constrained residue.

In some embodiments, the engineered transaminase having the features atone or more residue positions X69, X122, X223, and X284 as describedabove, includes at least the following additional features: X26 is P, H,F, or W, particularly H, and/or X62 is S, T, N, Q, Y, F, or W,particularly T or F; X65 is A, L or I, particularly A; X136 is Y, F orW, particularly Y or F; X199 is A, L, I, Y, F, or W, particularly W orI; and X209 is V, L or I, particularly L.

In some embodiments, the engineered transaminase having the features atone or more residue positions X69, X122, X223, and X284 as describedabove, includes at least the following additional features: X61 is Y, F,or W, particularly Y; X62 is S, T, N, Q, Y, F, or W, particularly T orF; X65 is A, L or I, particularly A; X94 is A, V, L or I, particularly Ior L; X136 is Y, F, or W, particularly Y or F; X199 is A, L, I, Y, F, orW, particularly W or I; X209 is V, L or I, particularly L; X215 is C;and X282 is S, N or Q, particularly S.

In some embodiments, the engineered transaminase having the features atone or more residue positions X69, X122, X223, and X284 as describedabove, includes at least the following additional features: X8 is H orP, particularly P; X61 is Y, F, or W, particularly Y; X62 is S, T, N Q,Y, F, or W, particularly T or F; X65 is A, L or I, particularly A; X81is G, M, A, V, L or I, particularly G; X94 is A, V, L or I, particularlyI or L; X136 is Y, F, or W, particularly Y or F; X199 is A, L, I, Y, F,or W, particularly W or I; X209 is V, L or I, particularly L; X215 is C;X217 is S, T, N, or Q, particularly N; X269 is H or P, particularly P;X282 is S, N or Q, particularly S. X297 is S, T, N or Q, particularly S;and X321 is H or P, particularly P.

In some embodiments, the engineered transaminase having the features atone or more residue positions X69, X122, X223, and X284 as describedabove, includes at least the following additional features: X8 is H orP, particularly P; X60 is F or W, particularly F; X61 is Y, F, or W,particularly Y; X62 is Y, F, W, S, T, N or Q, particularly T or F; X65is A, L or I, particularly A; X81 is G, M, A, V, L or I, particularly G;X94 is A, V, L or I, particularly I or L; X96 is A, V or L, particularlyL; X124 is P, H, T, N, or Q, particularly T, H or N; X136 is Y, F or W,particularly Y or F; X169 is V, L, or I, particularly L; X199 is Y, F,W, A, L or I, particularly W or I; X209 is V, L or I, particularly L;X215 is C; X217 is S, T, N or Q, particularly N; X269 is H or P,particularly P; X273 is Y, F, or W, particularly Y; X282 is S, N or Q,particularly S; X297 is S, T, N or Q, particularly S; and X321 is H orP, particularly P.

In some embodiments, the engineered transaminase having the features atone or more residue positions X69, X122, X223, and X284 as describedabove, includes at least the following additional features: X8 is H orP, particularly P; X60 is F or W, particularly F; X61 is Y, F, or W,particularly Y; X62 is Y, F, W, S, T, N or Q, particularly T or F; X65is A, L or I, particularly A; X81 is G, M, A, V, L or I, particularly G;X94 is A, V, L or I, particularly I or L; X96 is A, V or L, particularlyL; X124 is P, H, T, N, or Q, particularly T, H or N; X 126 is N, Q, orT, particularly T; X136 is Y, F or W, particularly Y or F; X150 is F, W,H, P, S, T, N, or Q, particularly F, H, or S; X152 is C, G, M, A, L, I,S, T, N, or Q, particularly G, I, L, S or C; X169 is V, L, or I,particularly L; X199 is Y, F, W, A, L or I, particularly W or I; X209 isV, L or I, particularly L; X215 is C; X217 is S, T, N or Q, particularlyN; X269 is H or P, particularly P; X273 is Y, F, or W, particularly Y;X282 is S, N or Q, particularly S; X297 is S, T, N or Q, particularly S;and X321 is H or P, particularly P.

In some embodiments, the engineered transaminases comprise an amino acidsequence that includes at least the following features: X122 is aconstrained, non-polar or aliphatic residue, particularly M, I, L, V, orH; X223 is a constrained residue, particularly P; X284 is a non-polarresidue, particularly G. In some embodiments, the transaminasepolypeptides can have additionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8,1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26,1-30, 1-35, 1-40, 1-45, 1-50, 1-55, or 1-60 residue differences at otherresidue positions. In some embodiments, the number of differences can be1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26,30, 35, 40, 45, 50, 55, or 60 residue differences at the other residuepositions. In some embodiments, the engineered transaminase polypeptidecan comprise an amino acid sequence that is at least about 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical to a reference amino acid sequence based on SEQ ID NO:2 havingthe features described for the preceding specified residue positions(i.e. X122; X223; and X284) (e.g., SEQ ID NO:8 or 10), with the provisothat the engineered transaminase polypeptide comprises polypeptidecomprises an amino acid sequence that includes at least the featuresdescribed for the specified residues.

In some embodiments, the engineered transaminases comprise an amino acidsequence that includes at least the following features: X69 is C or anon-polar, aliphatic or polar residue, particularly G, C, T, A, or S;X122 is a constrained, non-polar or aliphatic residue, particularly M,I, L, V, or H; X223 is a constrained residue, particularly P; and X284is a non-polar residue, particularly G. In some embodiments, thetransaminase polypeptides can have additionally 1-2, 1-3, 1-4, 1-5, 1-6,1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22,1-24, 1-26, 1-30, 1-35, 1-40, 1-45, 1-50, 1-55, or 1-60 residuedifferences at other residue positions. In some embodiments, the numberof differences can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16,18, 20, 22, 24, 26, 30, 35, 40, 45, 50, 55, or 60 residue differences atthe other residue positions. In some embodiments, the engineeredtransaminase polypeptide can comprise an amino acid sequence that is atleast about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% identical to a reference sequence based on SEQ IDNO:2 having the features described for preceding specified residuepositions (i.e., X69; X122; X223; and X284)(e.g., SEQ ID NO:4), with theproviso that the engineered transaminase polypeptide comprisespolypeptide comprises an amino acid sequence having at least thefeatures described for the specified residues.

In some embodiments, the engineered transaminases comprise an amino acidsequence that includes at least the following features: X65 is analiphatic residue, particularly A; X69 is C or a non-polar, aliphatic orpolar residue, particularly G, C, T, A, or S; X122 is a constrained,non-polar or aliphatic residue, particularly M, I, L, V, or H; and X223is a constrained residue, particularly P. In some embodiments, thetransaminase polypeptides can have additionally 1-2, 1-3, 1-4, 1-5, 1-6,1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22,1-24, 1-26, 1-30, 1-35, 1-40, 1-45, 1-50, 1-55, or 1-60 residuedifferences at other residue positions. In some embodiments, the numberof differences can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16,18, 20, 22, 24, 26, 30, 35, 40, 45, 50, 55, or 60 residue differences atthe other residue positions. In some embodiments, the engineeredtransaminase polypeptide can comprise an amino acid sequence that is atleast about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% identical to a reference sequence based on SEQ IDNO:2 having the features described for the preceding specified residuepositions (e.g., SEQ ID NO:6), with the proviso that the engineeredtransaminase polypeptide comprises polypeptide comprises an amino acidsequence that includes at least the features described for the specifiedresidues. In some embodiments, the engineered transaminase polypeptidecan comprise an amino acid sequence that is at least about 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical to a reference sequence of SEQ ID NO:6.

In some embodiments, the engineered transaminases comprise an amino acidsequence that includes at least the following features: X122 is aconstrained, non-polar or aliphatic residue, particularly M, I, L, V, orH; X174 is an aliphatic residue, particularly A; X223 is a constrainedresidue, particularly P; and X284 is a non-polar residue, particularlyG. In some embodiments, the transaminase polypeptides can haveadditionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12,1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35, 1-40, 1-45,1-50, 1-55, or 1-60 residue differences at other residue positions. Insome embodiments, the number of differences can be 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35, 40, 45, 50,55, or 60 residue differences at the other residue positions. In someembodiments, the engineered transaminase polypeptide can comprise anamino acid sequence that is at least about 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to areference sequence based on SEQ ID NO:2 having the features describedfor preceding specified residue positions (e.g., SEQ ID NO:12), with theproviso that the engineered transaminase polypeptide comprisespolypeptide comprises an amino acid sequence that includes at least thefeatures described for the specified residue positions. In someembodiments, the engineered transaminase polypeptide can comprise anamino acid sequence that is at least about 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to areference sequence of SEQ ID NO:12.

In some embodiments, the engineered transaminases comprise an amino acidsequence that includes at least the following features: X26 is anaromatic or constrained residue, particularly H; X65 is an aliphaticresidue, particularly A; X69 is C or a non-polar, aliphatic or polarresidue, particularly G, C, T, A, or S; X122 is a constrained, non-polaror aliphatic residue, particularly M, I, L, V, or H; X223 is aconstrained residue, particularly P; and X284 is a non-polar residue,particularly G. In some embodiments, the transaminase polypeptides canhave additionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11,1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35, 1-40,1-45, 1-50, 1-55, or 1-60 residue differences at other residuepositions. In some embodiments, the number of differences can be 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35,40, 45, 50, 55, or 60 residue differences at the other residuepositions. In some embodiments, the engineered transaminase polypeptidecan comprise an amino acid sequence that is at least about 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical to a reference sequence based on SEQ ID NO:2 having thefeatures described for preceding specified residue positions (e.g., SEQID NO:14), with the proviso that the engineered transaminase polypeptidecomprises polypeptide comprises an amino acid sequence that includes atleast the features described for the specified residue positions. Insome embodiments, the engineered transaminase polypeptide can comprisean amino acid sequence that is at least about 80%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to areference sequence of SEQ ID NO:14.

In some embodiments, the engineered transaminases comprise an amino acidsequence that includes at least the following features: X26 is anaromatic or constrained residue, particularly H; X62 is an aromatic orpolar residue, particularly T, Y or F; X65 is an aliphatic residue,particularly A; X69 is C or a non-polar, aliphatic or polar residue,particularly G, C, T, A, or S; X122 is a constrained, non-polar oraliphatic residue, particularly M, I, L, V, or H; X178 is a polarresidue, particularly S; X199 is an aliphatic or aromatic residue,particularly W or I, particularly X223 is a constrained residue,particularly P; X225 is an aromatic residue, particularly Y, X282 is apolar residue, particularly S; and X284 is a non-polar residue,particularly G. In some embodiments, the transaminase polypeptides canhave additionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11,1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35, 1-40,1-45, 1-50, 1-55, or 1-60 residue differences at other residuepositions. In some embodiments, the number of differences can be 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35,40, 45, 50, 55, or 60 residue differences at the other residuepositions. In some embodiments, the engineered transaminase polypeptidecan comprise an amino acid sequence that is at least about 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical to a reference sequence based on SEQ ID NO:2 having thefeatures described for preceding specified residue positions (e.g., SEQID NO:16), with the proviso that the engineered transaminase polypeptidecomprises polypeptide comprises an amino acid sequence that includes atleast the features described for the specified residue positions. Insome embodiments, the engineered transaminase polypeptide can comprisean amino acid sequence that is at least about 80%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to areference sequence of SEQ ID NO:16.

In some embodiments, the engineered transaminases comprise an amino acidsequence that includes at least the following features: X26 is anaromatic or constrained residue, particularly H; X62 is an aromatic orpolar residue, particularly T, Y or F; X65 is an aliphatic residue,particularly A; X69 is C or a non-polar, aliphatic or polar residue,particularly G, C, T, A, or S; X122 is a constrained, non-polar oraliphatic residue, particularly M, I, L, V, or H; X136 is an aromaticresidue, particularly Y or F; X199 is an aliphatic or aromatic residue,particularly W or I; X209 is an aliphatic residue, particularly L; X223is a constrained residue, particularly P; X225 is an aromatic residue,particularly Y, X282 is a polar residue, particularly S; and X284 is anon-polar residue, particularly G. In some embodiments, the transaminasepolypeptides can have additionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8,1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26,1-30, 1-35, 1-40, 1-45, 1-50, 1-55, or 1-60 residue differences at otherresidue positions. In some embodiments, the number of differences can be1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26,30, 35, 40, 45, 50, 55, or 60 residue differences at the other residuepositions. In some embodiments, the engineered transaminase polypeptidecan comprise an amino acid sequence that is at least about 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical to a reference sequence based on SEQ ID NO:2 having thefeatures described for preceding specified residue positions (e.g., SEQID NO:18), with the proviso that the engineered transaminase polypeptidecomprises polypeptide comprises an amino acid sequence that includes atleast the features described for the specified residue positions. Insome embodiments, the engineered transaminase polypeptide can comprisean amino acid sequence that is at least about 80%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to areference sequence of SEQ ID NO:18.

In some embodiments, the engineered transaminases comprise an amino acidsequence that includes at least the following features: X26 is anaromatic or constrained residue, particularly H; X62 is an aromatic orpolar residue, particularly T, Y or F; X65 is an aliphatic residue,particularly A; X69 is C or a non-polar, aliphatic or polar residue,particularly G, C, T, A, or S; X122 is a constrained, non-polar oraliphatic residue, particularly M, I, L, V, or H; X136 is an aromaticresidue, particularly Y or F; X137 is a polar or aliphatic residue,particularly T or I; X199 is an aliphatic or aromatic residue,particularly W or I; X209 is an aliphatic residue, particularly L; X223is a constrained residue, particularly P; X282 is a polar residue,particularly S; and X284 is a non-polar residue, particularly G. In someembodiments, the transaminase polypeptides can have additionally 1-2,1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16,1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35, 1-40, 1-45, 1-50, 1-55, or1-60 residue differences at other residue positions. In someembodiments, the number of differences can be 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35, 40, 45, 50, 55, or60 residue differences at the other residue positions. In someembodiments, the engineered transaminase polypeptide can comprise anamino acid sequence that is at least about 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to areference sequence based on SEQ ID NO:2 having the features describedfor preceding specified residue positions (e.g., SEQ ID NO:20, 22, 28,30, 32, 34, 38 or 40), with the proviso that the engineered transaminasepolypeptide comprises polypeptide comprises an amino acid sequence thatincludes at least the features described for the specified residuepositions. In some embodiments, the engineered transaminase polypeptidecan comprise an amino acid sequence that is at least about 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical to a reference sequence of SEQ ID NO:20, 22, 28, 30, 32, 34,38 or 40.

In some embodiments, the engineered transaminases comprise an amino acidsequence that includes at least the following features: X26 is anaromatic or constrained residue, particularly H; X62 is an aromatic orpolar residue, particularly T, Y or F; X65 is an aliphatic residue,particularly A; X69 is C or a non-polar, aliphatic or polar residue,particularly G, C, T, A, or S; X122 is a constrained, non-polar oraliphatic residue, particularly M, I, L, V, or H; X136 is an aromaticresidue, particularly Y or F; X137 is a polar or aliphatic residue,particularly T or I; X199 is an aliphatic or aromatic residue,particularly W or I; X209 is an aliphatic residue, particularly L; X223is a constrained residue, particularly P; X225 is an aromatic residue,particularly Y, X282 is a polar residue, particularly S; and X284 is anon-polar residue, particularly G. In some embodiments, the transaminasepolypeptides can have additionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8,1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26,1-30, 1-35, 1-40, 1-45, 1-50, 1-55, or 1-60 residue differences at otherresidue positions. In some embodiments, the number of differences can be1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26,30, 35, 40, 45, 50, 55, or 60 residue differences at the other residuepositions. In some embodiments, the engineered transaminase polypeptidecan comprise an amino acid sequence that is at least about 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical to a reference sequence based on SEQ ID NO:2 having thefeatures described for preceding specified residue positions (e.g., SEQID NO:24), with the proviso that the engineered transaminase polypeptidecomprises polypeptide comprises an amino acid sequence that includes atleast the features described for the specified residue positions. Insome embodiments, the engineered transaminase polypeptide can comprisean amino acid sequence that is at least about 80%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to areference sequence of SEQ ID NO:24.

In some embodiments, the engineered transaminases comprise an amino acidsequence that includes at least the following features: X26 is anaromatic or constrained residue, particularly H; X65 is an aliphaticresidue, particularly A; X69 is C or a non-polar, aliphatic or polarresidue, particularly G, C, T, A, or S; X122 is a constrained, non-polaror aliphatic residue, particularly M, I, L, V, or H; X136 is an aromaticresidue, particularly Y or F; X137 is a polar or aliphatic residue,particularly T or I; X174 is an aliphatic residue, particularly A; X199is an aliphatic or aromatic residue, particularly W or I; X209 is analiphatic residue, particularly L; X223 is a constrained residue,particularly P; X230 is an aliphatic residue, particularly V; and X284is a non-polar residue, particularly G. In some embodiments, thetransaminase polypeptides can have additionally 1-2, 1-3, 1-4, 1-5, 1-6,1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22,1-24, 1-26, 1-30, 1-35, 1-40, 1-45, 1-50, 1-55, or 1-60 residuedifferences at other residue positions. In some embodiments, the numberof differences can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16,18, 20, 22, 24, 26, 30, 35, 40, 45, 50, 55, or 60 residue differences atthe other residue positions. In some embodiments, the engineeredtransaminase polypeptide can comprise an amino acid sequence that is atleast about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% identical to a reference sequence based on SEQ IDNO:2 having the features described for preceding specified residuepositions (e.g., SEQ ID NO:26), with the proviso that the engineeredtransaminase polypeptide comprises polypeptide comprises an amino acidsequence that includes at least the features described for the specifiedresidue positions. In some embodiments, the engineered transaminasepolypeptide can comprise an amino acid sequence that is at least about80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% identical to a reference sequence of SEQ ID NO:26.

In some embodiments, the engineered transaminases comprise an amino acidsequence that includes at least the following features: X26 is anaromatic or constrained residue, particularly H; X61 is an aromaticresidue, particularly Y; X62 is an aromatic or polar residue,particularly T, Y or F; X65 is an aliphatic residue, particularly A; X69is C or a non-polar, aliphatic or polar residue, particularly G, C, T,A, or S; X122 is a constrained, non-polar or aliphatic residue,particularly M, I, L, V, or H; X136 is an aromatic residue, particularlyY or F; X137 is a polar or aliphatic residue, particularly T or I; X199is an aliphatic or aromatic residue, particularly W or I; X209 is analiphatic residue, particularly L; X223 is a constrained residue,particularly P; X282 is a polar residue, particularly S; and X284 is anon-polar residue, particularly G. In some embodiments, the transaminasepolypeptides can have additionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8,1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26,1-30, 1-35, 1-40, 1-45, 1-50, 1-55, or 1-60 residue differences at otherresidue positions. In some embodiments, the number of differences can be1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26,30, 35, 40, 45, 50, 55, or 60 residue differences at the other residuepositions. In some embodiments, the engineered transaminase polypeptidecan comprise an amino acid sequence that is at least about 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical to a reference sequence based on SEQ ID NO:2 having thefeatures described for preceding specified residue positions (e.g., SEQID NO:36), with the proviso that the engineered transaminase polypeptidecomprises polypeptide comprises an amino acid sequence that includes atleast the features described for the specified residue positions. Insome embodiments, the engineered transaminase polypeptide can comprisean amino acid sequence that is at least about 80%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to areference sequence of SEQ ID NO:36.

In some embodiments, the engineered transaminases comprise an amino acidsequence that includes at least the following features: X4 is anaromatic residue, particularly Y; X26 is an aromatic or constrainedresidue, particularly H; X62 is an aromatic or polar residue,particularly T, Y or F; X65 is an aliphatic residue, particularly A; X69is C or a non-polar, aliphatic or polar residue, particularly G, C, T,A, or S; X94 is an aliphatic residue, particularly I or L; X122 is aconstrained, non-polar or aliphatic residue, particularly M, I, L, V, orH; X136 is an aromatic residue, particularly Y or F; X137 is a polar oraliphatic residue, particularly T or I; X199 is an aliphatic or aromaticresidue, particularly W or I; X209 is an aliphatic residue, particularlyL; X215 is C; X223 is a constrained residue, particularly P; X282 is apolar residue, particularly S; and X284 is a non-polar residue,particularly G. In some embodiments, the transaminase polypeptides canhave additionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11,1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35, 1-40,1-45, 1-50, 1-55, or 1-60 residue differences at other residuepositions. In some embodiments, the number of differences can be 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35,40, 45, 50, 55, or 60 residue differences at the other residuepositions. In some embodiments, the engineered transaminase polypeptidecan comprise an amino acid sequence that is at least about 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical to a reference sequence based on SEQ ID NO:2 having thefeatures described for preceding specified residue positions (e.g., SEQID NO:42), with the proviso that the engineered transaminase polypeptidecomprises polypeptide comprises an amino acid sequence that includes atleast the features described for the specified residue positions. Insome embodiments, the engineered transaminase polypeptide can comprisean amino acid sequence that is at least about 80%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to areference sequence of SEQ ID NO:42.

In some embodiments, the engineered transaminase comprises an amino acidsequence that includes the following features: X62 is an aromatic orpolar residue, particularly T, Y or F; X65 is an aliphatic residue,particularly A; X69 is C or a non-polar, aliphatic or polar residue,particularly G, C, T, A, or S; X94 is an aliphatic residue, particularlyI or L; X122 is a constrained, non-polar or aliphatic residue,particularly M, I, L, V, or H; X136 is an aromatic residue, particularlyY or F; X137 is a polar or aliphatic residue, particularly T or I; X199is an aliphatic or aromatic residue, particularly W or I; X209 is analiphatic residue, particularly L; X215 is a C; X223 is a constrainedresidue, particularly P; X282 is a polar residue, particularly S; andX284 is a non-polar residue, particularly G. In some embodiments, thetransaminase polypeptides can have additionally 1-2, 1-3, 1-4, 1-5, 1-6,1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22,1-24, 1-26, 1-30, 1-35, 1-40, 1-45, 1-50, 1-55, or 1-60 residuedifferences at other residue positions. In some embodiments, the numberof differences can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16,18, 20, 22, 24, 26, 30, 35, 40, 45, 50, 55, or 60 residue differences atthe other residue positions. In some embodiments, the engineeredtransaminase polypeptide can comprise an amino acid sequence that is atleast about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% identical to a reference sequence based on SEQ IDNO:2 having the features described for preceding specified residuepositions (e.g., SEQ ID NO: 44, 46, or 48), with the proviso that theengineered transaminase polypeptide comprises polypeptide comprises anamino acid sequence that includes at least the features described forthe specified residue positions. In some embodiments, the engineeredtransaminase polypeptide can comprise an amino acid sequence that is atleast about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% identical to a reference sequence of SEQ ID NO:44,46, or 48.

In some embodiments, the engineered transaminase comprises an amino acidsequence that includes the following features: X8 is a constrainedresidue, particularly P; X62 is an aromatic or polar residue,particularly T, Y or F; X65 is an aliphatic residue, particularly A; X69is C or a non-polar, aliphatic or polar residue, particularly G, C, T,A, or S; X94 is an aliphatic residue, particularly I or L; X122 is aconstrained, non-polar or aliphatic residue, particularly M, I, L, V, orH; X136 is an aromatic residue, particularly Y or F; X137 is a polar oraliphatic residue, particularly T or I; X199 is an aliphatic or aromaticresidue, particularly W or I; X209 is an aliphatic residue, particularlyL; X215 is a cysteine (C); X223 is a constrained residue, particularlyP; X282 is a polar residue, particularly S; and X284 is a non-polarresidue, particularly G. In some embodiments, the transaminasepolypeptides can have additionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8,1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26,1-30, 1-35, 1-40, 1-45, 1-50, 1-55, or 1-60 residue differences at otherresidue positions. In some embodiments, the number of differences can be1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26,30, 35, 40, 45, 50, 55, or 60 residue differences at the other residuepositions. In some embodiments, the engineered transaminase polypeptidecan comprise an amino acid sequence that is at least about 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical to a reference sequence based on SEQ ID NO:2 having thefeatures described for preceding specified residue positions (e.g., SEQID NO: 50), with the proviso that the engineered transaminasepolypeptide comprises polypeptide comprises an amino acid sequence thatincludes at least the features described for the specified residuepositions. In some embodiments, the engineered transaminase polypeptidecan comprise an amino acid sequence that is at least about 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical to a reference sequence of SEQ ID NO:50.

In some embodiments, the engineered transaminase comprises an amino acidsequence that includes the following features: X61 is an aromaticresidue, particularly Y; X62 is an aromatic or polar residue,particularly T, Y or F; X65 is an aliphatic residue, particularly A; X69is C or a non-polar, aliphatic or polar residue, particularly G, C, T,A, or S; X94 is an aliphatic residue, particularly I or L; X122 is aconstrained, non-polar or aliphatic residue, particularly M, I, L, V, orH; X136 is an aromatic residue, particularly Y or F; X137 is a polar oraliphatic residue, particularly T or I; X152 is C, non-polar, aliphatic,or polar residue, particularly G, I, L, S or C; X199 is an aliphatic oraromatic residue, particularly W or I; X209 is an aliphatic residue,particularly L; X215 is a C; X223 is a constrained residue, particularlyP; X282 is a polar residue, particularly S; and X284 is a non-polarresidue, particularly G. In some embodiments, the transaminasepolypeptides can have additionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8,1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26,1-30, 1-35, 1-40, 1-45, 1-50, 1-55, or 1-60 residue differences at otherresidue positions. In some embodiments, the number of differences can be1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26,30, 35, 40, 45, 50, 55, or 60 residue differences at the other residuepositions. In some embodiments, the engineered transaminase polypeptidecan comprise an amino acid sequence that is at least about 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical to a reference sequence based on SEQ ID NO:2 having thefeatures described for preceding specified residue positions (e.g., SEQID NO: 52), with the proviso that the engineered transaminasepolypeptide comprises polypeptide comprises an amino acid sequence thatincludes at least the features described for the specified residuepositions. In some embodiments, the engineered transaminase polypeptidecan comprise an amino acid sequence that is at least about 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical to a reference sequence of SEQ ID NO:52.

In some embodiments, the engineered transaminase comprises an amino acidsequence that includes the following features: X61 is an aromaticresidue, particularly Y; X62 is an aromatic or polar residue,particularly T, Y or F; X65 is an aliphatic residue, particularly A; X69is C or a non-polar, aliphatic or polar residue, particularly G, C, T,A, or S; X94 is an aliphatic residue, particularly I or L; X122 is aconstrained, non-polar or aliphatic residue, particularly M, I, L, V, orH; X136 is an aromatic residue, particularly Y or F; X137 is a polar oraliphatic residue, particularly T or I; X199 is an aliphatic or aromaticresidue, particularly W or I; X209 is an aliphatic residue, particularlyL; X215 is a C; X223 is a constrained residue, particularly P; X282 is apolar residue, particularly S; and X284 is a non-polar residue,particularly G. In some embodiments, the transaminase polypeptides canhave additionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11,1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35, 1-40,1-45, 1-50, 1-55, or 1-60 residue differences at other residuepositions. In some embodiments, the number of differences can be 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35,40, 45, 50, 55, or 60 residue differences at the other residuepositions. In some embodiments, the engineered transaminase polypeptidecan comprise an amino acid sequence that is at least about 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical to a reference sequence based on SEQ ID NO:2 having thefeatures described for preceding specified residue positions (e.g., SEQID NO: 54 or 56), with the proviso that the engineered transaminasepolypeptide comprises polypeptide comprises an amino acid sequence thatincludes at least the features described for the specified residuepositions. In some embodiments, the engineered transaminase polypeptidecan comprise an amino acid sequence that is at least about 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical to a reference sequence of SEQ ID NO:54 or 56.

In some embodiments, the engineered transaminase comprises an amino acidsequence that includes the following features: X61 is an aromaticresidue, particularly Y; X62 is an aromatic or polar residue,particularly T, Y or F; X65 is an aliphatic residue, particularly A; X69is C or a non-polar, aliphatic or polar residue, particularly G, C, T,A, or S; X94 is an aliphatic residue, particularly I or L; X122 is aconstrained, non-polar or aliphatic residue, particularly M, I, L, V, orH; X136 is an aromatic residue, particularly Y or F; X199 is analiphatic or aromatic residue, particularly W or I; X209 is an aliphaticresidue, particularly L; X215 is a C; X223 is a constrained residue,particularly P; X282 is a polar residue, particularly S; and X284 is anon-polar residue, particularly G. In some embodiments, the transaminasepolypeptides can have additionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8,1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26,1-30, 1-35, 1-40, 1-45, 1-50, 1-55, or 1-60 residue differences at otherresidue positions. In some embodiments, the number of differences can be1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26,30, 35, 40, 45, 50, 55, or 60 residue differences at the other residuepositions. In some embodiments, the engineered transaminase polypeptidecan comprise an amino acid sequence that is at least about 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical to a reference sequence based on SEQ ID NO:2 having thefeatures described for preceding specified residue positions (e.g., SEQID NO: 58 or 60), with the proviso that the engineered transaminasepolypeptide comprises polypeptide comprises an amino acid sequence thatincludes at least the features described for the specified residuepositions. In some embodiments, the engineered transaminase polypeptidecan comprise an amino acid sequence that is at least about 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical to a reference sequence of SEQ ID NO:58 or 60.

In some embodiments, the engineered transaminase comprises an amino acidsequence that includes the following features: X61 is an aromaticresidue, particularly Y; X62 is an aromatic or polar residue,particularly T, Y or F; X65 is an aliphatic residue, particularly A; X69is C or a non-polar, aliphatic or polar residue, particularly G, C, T,A, or S; X94 is an aliphatic residue, particularly I or L; X122 is aconstrained, non-polar or aliphatic residue, particularly M, I, L, V, orH; X136 is an aromatic residue, particularly Y or F; X137 is a polar oraliphatic residue, particularly T or I; X160 is an aliphatic residue,particularly L; X169 is an aliphatic residue, particularly L; X199 is analiphatic or aromatic residue, particularly W or I; X209 is an aliphaticresidue, particularly L; X215 is C; X223 is a constrained residue,particularly P; X269 is a constrained residue, particularly P; X282 is apolar residue, particularly S; and X284 is a non-polar residue,particularly G. In some embodiments, the transaminase polypeptides canhave additionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11,1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35, 1-40,1-45, 1-50, 1-55, or 1-60 residue differences at other residuepositions. In some embodiments, the number of differences can be 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35,40, 45, 50, 55, or 60 residue differences at the other residuepositions. In some embodiments, the engineered transaminase polypeptidecan comprise an amino acid sequence that is at least about 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical to a reference sequence based on SEQ ID NO:2 having thefeatures described for preceding specified residue positions (e.g., SEQID NO:62), with the proviso that the engineered transaminase polypeptidecomprises polypeptide comprises an amino acid sequence that includes atleast the features described for the specified residue positions. Insome embodiments, the engineered transaminase polypeptide can comprisean amino acid sequence that is at least about 80%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to areference sequence of SEQ ID NO:62.

In some embodiments, the engineered transaminase comprises an amino acidsequence that includes the following features: X61 is an aromaticresidue, particularly Y; X62 is an aromatic or polar residue,particularly T, Y or F; X65 is an aliphatic residue, particularly A; X69is C or a non-polar, aliphatic or polar residue, particularly G, C, T,A, or S; X94 is an aliphatic residue, particularly I or L; X122 is aconstrained, non-polar or aliphatic residue, particularly M, I, L, V, orH; X136 is an aromatic residue, particularly Y or F; X137 is a polar oraliphatic residue, particularly T or I; X169 is an aliphatic residue,particularly L; X199 is an aliphatic or aromatic residue, particularly Wor I; X209 is an aliphatic residue, particularly L; X215 is C; X223 is aconstrained residue, particularly P; X282 is a polar residue,particularly S; X284 is a non-polar residue, particularly G; and X306 isan aliphatic residue, particularly L. In some embodiments, thetransaminase polypeptides can have additionally 1-2, 1-3, 1-4, 1-5, 1-6,1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22,1-24, 1-26, 1-30, 1-35, 1-40, 1-45, 1-50, 1-55, or 1-60 residuedifferences at other residue positions. In some embodiments, the numberof differences can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16,18, 20, 22, 24, 26, 30, 35, 40, 45, 50, 55, or 60 residue differences atthe other residue positions. In some embodiments, the engineeredtransaminase polypeptide can comprise an amino acid sequence that is atleast about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% identical to a reference sequence based on SEQ IDNO:2 having the features described for preceding specified residuepositions (e.g., SEQ ID NO:64), with the proviso that the engineeredtransaminase polypeptide comprises polypeptide comprises an amino acidsequence that includes at least the features described for the specifiedresidue positions. In some embodiments, the engineered transaminasepolypeptide can comprise an amino acid sequence that is at least about80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% identical to a reference sequence of SEQ ID NO:64.

In some embodiments, the engineered transaminase comprises an amino acidsequence that includes the following features: X61 is an aromaticresidue, particularly Y; X62 is an aromatic or polar residue,particularly T, Y or F; X65 is an aliphatic residue, particularly A; X69is C or a non-polar, aliphatic or polar residue, particularly G, C, T,A, or S; X94 is an aliphatic residue, particularly I or L; X102 is analiphatic or basic residue, particularly L or K; X122 is a constrained,non-polar or aliphatic residue, particularly M, I, L, V, or H; X136 isan aromatic residue, particularly Y or F; X150 is aromatic, constrainedor polar residue, particularly F, H, or S; X152 is C, non-polar,aliphatic, or polar residue, particularly G, I, L, S or C; X199 is analiphatic or aromatic residue, particularly W or I; X209 is an aliphaticresidue, particularly L; X215 is a C; X223 is a constrained residue,particularly P; X282 is a polar residue, particularly S; and X284 is anon-polar residue, particularly G. In some embodiments, the transaminasepolypeptides can have additionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8,1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26,1-30, 1-35, 1-40, 1-45, 1-50, 1-55, or 1-60 residue differences at otherresidue positions. In some embodiments, the number of differences can be1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26,30, 35, 40, 45, 50, 55, or 60 residue differences at the other residuepositions. In some embodiments, the engineered transaminase polypeptidecan comprise an amino acid sequence that is at least about 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical to a reference sequence based on SEQ ID NO:2 having thefeatures described for preceding specified residue positions (e.g., SEQID NO:66), with the proviso that the engineered transaminase polypeptidecomprises polypeptide comprises an amino acid sequence that includes atleast the features described for the specified residue positions. Insome embodiments, the engineered transaminase polypeptide can comprisean amino acid sequence that is at least about 80%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to areference sequence of SEQ ID NO:66.

In some embodiments, the engineered transaminase comprises an amino acidsequence that includes the following features: X8 is a constrainedresidue, particularly is P; X48 is a polar, acidic aliphatic ornon-polar residue, particularly D, V, G, Q or A; X61 is an aromaticresidue, particularly Y; X62 is an aromatic or polar residue,particularly T, Y or F; X65 is an aliphatic residue, particularly A; X69is C or a non-polar, aliphatic or polar residue, particularly G, C, T,A, or S; X81 is a non-polar residue, particularly G; X94 is an aliphaticresidue, particularly I or L; X96 is an aliphatic residue, particularlyL; X102 is an aliphatic or basic residue, particularly L or K; X122 is aconstrained, non-polar or aliphatic residue, particularly M, I, L, V, orH; X136 is an aromatic residue, particularly Y or F; X163 is analiphatic or constrained residue, particularly H or V; X199 is analiphatic or aromatic residue, particularly W or I; X209 is an aliphaticresidue, particularly L; X211 is an aliphatic residue, particularly I;X215 is a C; X217 is a polar residue, particularly N; X223 is aconstrained residue, particularly P; X252 is an aromatic or aliphaticresidue, particularly F; X273 is an aromatic residue, particularly Y;X282 is a polar residue, particularly S; and X284 is a non-polarresidue, particularly G; and X321 is a constrained residue, particularlyP. In some embodiments, the transaminase polypeptides can haveadditionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12,1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35, 1-40, 1-45,1-50, 1-55, or 1-60 residue differences at other residue positions. Insome embodiments, the number of differences can be 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35, 40, 45, 50,55, or 60 residue differences at the other residue positions. In someembodiments, the engineered transaminase polypeptide can comprise anamino acid sequence that is at least about 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to areference sequence based on SEQ ID NO:2 having the features describedfor preceding specified residue positions (e.g., SEQ ID NO:68), with theproviso that the engineered transaminase polypeptide comprisespolypeptide comprises an amino acid sequence that includes at least thefeatures described for the specified residue positions. In someembodiments, the engineered transaminase polypeptide can comprise anamino acid sequence that is at least about 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to areference sequence of SEQ ID NO:68.

In some embodiments, the engineered transaminase comprises an amino acidsequence that includes the following features: X8 is a constrainedresidue, particularly is P; X48 is a polar, acidic, aliphatic ornon-polar residue, particularly A; X61 is an aromatic residue,particularly Y; X62 is an aromatic or polar residue, particularly T, Yor F; X65 is an aliphatic residue, particularly A; X69 is C or anon-polar, aliphatic or polar residue, particularly G, C, T, A, or S;X81 is a non-polar residue, particularly G; X94 is an aliphatic residue,particularly I or L; X122 is a constrained, non-polar or aliphaticresidue, particularly M, I, L, V, or H; X136 is an aromatic residue,particularly Y or F; X169 is an aliphatic residue, particularly L; X199is an aliphatic or aromatic residue, particularly W or I; X209 is analiphatic residue, particularly L; X215 is C; X217 is a polar residue,particularly N; X223 is a constrained residue, particularly P; X269 is aconstrained residue, particularly P; X282 is a polar residue,particularly S; X284 is a non-polar residue, particularly G; X297 is apolar residue, particularly S; and X321 is a constrained residue,particularly P. In some embodiments, the transaminase polypeptides canhave additionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11,1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35, 1-40,1-45, 1-50, 1-55, or 1-60 residue differences at other residuepositions. In some embodiments, the number of differences can be 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35,40, 45, 50, 55, or 60 residue differences at the other residuepositions. In some embodiments, the engineered transaminase polypeptidecan comprise an amino acid sequence that is at least about 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical to a reference sequence based on SEQ ID NO:2 having thefeatures described for preceding specified residue positions (e.g., SEQID NO:70), with the proviso that the engineered transaminase polypeptidecomprises an amino acid sequence that includes at least the featuresdescribed for the specified residue positions. In some embodiments, theengineered transaminase polypeptide can comprise an amino acid sequencethat is at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% identical to a reference sequence of SEQ IDNO:70.

In some embodiments, the engineered transaminase comprises an amino acidsequence that includes the following features: X8 is a constrainedresidue, particularly is P; X61 is an aromatic residue, particularly Y;X62 is an aromatic or polar residue, particularly T, Y or F; X65 is analiphatic residue, particularly A; X69 is C or a non-polar, aliphatic orpolar residue, particularly G, C, T, A, or S; X94 is an aliphaticresidue, particularly I or L; X122 is a constrained, non-polar oraliphatic residue, particularly M, I, L, V, or H; X136 is an aromaticresidue, particularly Y or F; X199 is an aliphatic or aromatic residue,particularly W or I; X209 is an aliphatic residue, particularly L; X215is C; X223 is a constrained residue, particularly P; X282 is a polarresidue, particularly S; and X284 is a non-polar residue, particularlyG. In some embodiments, the transaminase polypeptides can haveadditionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12,1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35, 1-40, 1-45,1-50, 1-55, or 1-60 residue differences at other residue positions. Insome embodiments, the number of differences can be 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35, 40, 45, 50,55, or 60 residue differences at the other residue positions. In someembodiments, the engineered transaminase polypeptide can comprise anamino acid sequence that is at least about 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to areference sequence based on SEQ ID NO:2 having the features describedfor preceding specified residue positions (e.g., SEQ ID NO:72), with theproviso that the engineered transaminase polypeptide comprises an aminoacid sequence that includes at least the features described for thespecified residue positions. In some embodiments, the engineeredtransaminase polypeptide can comprise an amino acid sequence that is atleast about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% identical to a reference sequence of SEQ ID NO:72.

In some embodiments, the engineered transaminase comprises an amino acidsequence that includes the following features: X8 is a constrainedresidue, particularly is P; X61 is an aromatic residue, particularly Y;X62 is an aromatic or polar residue, particularly T, Y or F; X65 is analiphatic residue, particularly A; X69 is C or a non-polar, aliphatic orpolar residue, particularly G, C, T, A, or S; X81 is a non-polarresidue, particularly G; X94 is an aliphatic residue, particularly I orL; X96 is an aliphatic residue, particularly L; X122 is a constrained,non-polar or aliphatic residue, particularly M, I, L, V, or H; X136 isan aromatic residue, particularly Y or F; X178 is a polar residue,particularly S; X199 is an aliphatic or aromatic residue, particularly Wor I; X209 is an aliphatic residue, particularly L; X215 is C; X223 is aconstrained residue, particularly P; X269 is a constrained residue,particularly P; X282 is a polar residue, particularly S; X284 is anon-polar residue, particularly G; X297 is a polar residue, particularlyS; and X321 is a constrained residue, particularly P. In someembodiments, the transaminase polypeptides can have additionally 1-2,1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16,1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35, 1-40, 1-45, 1-50, 1-55, or1-60 residue differences at other residue positions. In someembodiments, the number of differences can be 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35, 40, 45, 50, 55, or60 residue differences at the other residue positions. In someembodiments, the engineered transaminase polypeptide can comprise anamino acid sequence that is at least about 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to areference sequence based on SEQ ID NO:2 having the features describedfor preceding specified residue positions (e.g., SEQ ID NO:74), with theproviso that the engineered transaminase polypeptide comprises an aminoacid sequence that includes at least the features described for thespecified residue positions. In some embodiments, the engineeredtransaminase polypeptide can comprise an amino acid sequence that is atleast about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% identical to a reference sequence of SEQ ID NO:74.

In some embodiments, the engineered transaminase comprises an amino acidsequence that includes the following features: X8 is a constrainedresidue, particularly is P; X60 is an aromatic residue, particularly F;X61 is an aromatic residue, particularly Y; X62 is an aromatic or polarresidue, particularly T, Y or F; X65 is an aliphatic residue,particularly A; X69 is C or a non-polar, aliphatic or polar residue,particularly G, C, T, A, or S; X81 is a non-polar residue, particularlyG; X94 is an aliphatic residue, particularly I or L; X96 is an aliphaticresidue, particularly L; X122 is a constrained, non-polar or aliphaticresidue, particularly M, I, L, V, or H; X136 is an aromatic residue,particularly Y or F; X152 is C or a non-polar, aliphatic, or polarresidue, particularly G, I, L, S or C; X178 is a polar residue,particularly S; X199 is an aliphatic or aromatic residue, particularly Wor I; X209 is an aliphatic residue, particularly L; X215 is C; X217 is apolar residue, particularly N; X223 is a constrained residue,particularly P; X252 is an aromatic or aliphatic residue, particularlyF; X269 is a constrained residue, particularly P; X273 is an aromaticresidue, particularly Y; X282 is a polar residue, particularly S; andX284 is a non-polar residue, particularly G; X297 is a polar residue,particularly S; and X321 is a constrained residue, particularly P. Insome embodiments, the transaminase polypeptides can have additionally1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15,1-16, 1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35, 1-40, 1-45, 1-50, 1-55,or 1-60 residue differences at other residue positions. In someembodiments, the number of differences can be 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35, 40, 45, 50, 55, or60 residue differences at the other residue positions. In someembodiments, the engineered transaminase polypeptide can comprise anamino acid sequence that is at least about 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to areference sequence based on SEQ ID NO:2 having the features describedfor preceding specified residue positions (e.g., SEQ ID NO:76), with theproviso that the engineered transaminase polypeptide comprises an aminoacid sequence that includes at least the features described for thespecified residue positions. In some embodiments, the engineeredtransaminase polypeptide can comprise an amino acid sequence that is atleast about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% identical to a reference sequence of SEQ ID NO:76.

In some embodiments, the engineered transaminase comprises an amino acidsequence that includes the following features: X8 is a constrainedresidue, particularly is P; X60 is an aromatic residue, particularly F;X61 is an aromatic residue, particularly Y; X62 is an aromatic or polarresidue, particularly T, Y or F; X65 is an aliphatic residue,particularly A; X69 is C or a non-polar, aliphatic or polar residue,particularly G, C, T, A, or S; X81 is a non-polar residue, particularlyG; X94 is an aliphatic residue, particularly I or L; X96 is an aliphaticresidue, particularly L; X122 is a constrained, non-polar or aliphaticresidue, particularly M, I, L, V, or H; X136 is an aromatic residue,particularly Y or F; X169 is an aliphatic residue, particularly L; X178is a polar residue, particularly S; X199 is an aliphatic or aromaticresidue, particularly W or I; X209 is an aliphatic residue, particularlyL; X215 is C; X217 is a polar residue, particularly N; X223 is aconstrained residue, particularly P; X269 is a constrained residue,particularly P; X282 is a polar residue, particularly S; and X284 is anon-polar residue, particularly G; X292 is a polar residue, particularlyT; X297 is a polar residue, particularly S; and X321 is a constrainedresidue, particularly P. In some embodiments, the transaminasepolypeptides can have additionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8,1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26,1-30, 1-35, 1-40, 1-45, 1-50, 1-55, or 1-60 residue differences at otherresidue positions. In some embodiments, the number of differences can be1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26,30, 35, 40, 45, 50, 55, or 60 residue differences at the other residuepositions. In some embodiments, the engineered transaminase polypeptidecan comprise an amino acid sequence that is at least about 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical to a reference sequence based on SEQ ID NO:2 having thefeatures described for preceding specified residue positions (e.g., SEQID NO:78), with the proviso that the engineered transaminase polypeptidecomprises an amino acid sequence that includes at least the featuresdescribed for the specified residue positions. In some embodiments, theengineered transaminase polypeptide can comprise an amino acid sequencethat is at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% identical to a reference sequence of SEQ IDNO:78.

In some embodiments, the engineered transaminase comprises an amino acidsequence that includes the following features: X8 is a constrainedresidue, particularly is P; X60 is an aromatic residue, particularly F;X61 is an aromatic residue, particularly Y; X62 is an aromatic or polarresidue, particularly T, Y or F; X65 is an aliphatic residue,particularly A; X69 is C or a non-polar, aliphatic or polar residue,particularly G, C, T, A, or S; X81 is a non-polar residue, particularlyG; X94 is an aliphatic residue, particularly I or L; X96 is an aliphaticresidue, particularly L; X122 is a constrained, non-polar or aliphaticresidue, particularly M, I, L, V, or H; X136 is an aromatic residue,particularly Y or F; X169 is an aliphatic residue, particularly L; X199is an aliphatic or aromatic residue, particularly W or I; X209 is analiphatic residue, particularly L; X215 is C; X217 is a polar residue,particularly N; X223 is a constrained residue, particularly P; X269 is aconstrained residue, particularly P; X273 is an aromatic residue,particularly Y; X282 is a polar residue, particularly S; and X284 is anon-polar residue, particularly G; X297 is a polar residue, particularlyS; and X321 is a constrained residue, particularly P. In someembodiments, the transaminase polypeptides can have additionally 1-2,1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16,1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35, 1-40, 1-45, 1-50, 1-55, or1-60 residue differences at other residue positions. In someembodiments, the number of differences can be 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35, 40, 45, 50, 55, or60 residue differences at the other residue positions. In someembodiments, the engineered transaminase polypeptide can comprise anamino acid sequence that is at least about 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to areference sequence based on SEQ ID NO:2 having the features describedfor preceding specified residue positions (e.g., SEQ ID NO:80), with theproviso that the engineered transaminase polypeptide comprises an aminoacid sequence that includes at least the features described for thespecified residue positions. In some embodiments, the engineeredtransaminase polypeptide can comprise an amino acid sequence that is atleast about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% identical to a reference sequence of SEQ ID NO:80.

In some embodiments, the engineered transaminase comprises an amino acidsequence that includes the following features: X8 is a constrainedresidue, particularly is P; X60 is an aromatic residue, particularly F;X61 is an aromatic residue, particularly Y; X62 is an aromatic or polarresidue, particularly T, Y or F; X65 is an aliphatic residue,particularly A; X69 is C or a non-polar, aliphatic or polar residue,particularly G, C, T, A, or S; X81 is a non-polar residue, particularlyG; X94 is an aliphatic residue, particularly I or L; X96 is an aliphaticresidue, particularly L; X122 is a constrained, non-polar or aliphaticresidue, particularly M, I, L, V, or H; X136 is an aromatic residue,particularly Y or F; X169 is an aliphatic residue, particularly L; X178is a polar residue, particularly S; X199 is an aliphatic or aromaticresidue, particularly W or I; X209 is an aliphatic residue, particularlyL; X215 is C; X223 is a constrained residue, particularly P; X269 is aconstrained residue, particularly P; X273 is an aromatic residue,particularly Y; X282 is a polar residue, particularly S; and X284 is anon-polar residue, particularly G; X297 is a polar residue, particularlyS; and X321 is a constrained residue, particularly P. In someembodiments, the transaminase polypeptides can have additionally 1-2,1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16,1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35, 1-40, 1-45, 1-50, 1-55, or1-60 residue differences at other residue positions. In someembodiments, the number of differences can be 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35, 40, 45, 50, 55, or60 residue differences at the other residue positions. In someembodiments, the engineered transaminase polypeptide can comprise anamino acid sequence that is at least about 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to areference sequence based on SEQ ID NO:2 having the features describedfor preceding specified residue positions (e.g., SEQ ID NO:82), with theproviso that the engineered transaminase polypeptide comprises an aminoacid sequence that includes at least the features described for thespecified residue positions. In some embodiments, the engineeredtransaminase polypeptide can comprise an amino acid sequence that is atleast about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% identical to a reference sequence of SEQ ID NO:82

In some embodiments, the engineered transaminase comprises an amino acidsequence that includes the following features: X8 is a constrainedresidue, particularly is P; X60 is an aromatic residue, particularly F;X61 is an aromatic residue, particularly Y; X62 is an aromatic or polarresidue, particularly T, Y or F; X65 is an aliphatic residue,particularly A; X69 is C or a non-polar, aliphatic or polar residue,particularly G, C, T, A, or S; X81 is a non-polar residue, particularlyG; X94 is an aliphatic residue, particularly I or L; X96 is an aliphaticresidue, particularly L; X122 is a constrained, non-polar or aliphaticresidue, particularly M, I, L, V, or H; X124 is a polar or constrainedresidue, particularly T, H or N; X136 is an aromatic residue,particularly Y or F; X169 is an aliphatic residue, particularly L; X199is an aliphatic or aromatic residue, particularly W or I; X209 is analiphatic residue, particularly L; X215 is C; X217 is a polar residue,particularly N; X223 is a constrained residue, particularly P; X269 is aconstrained residue, particularly P; X273 is an aromatic residue,particularly Y; X282 is a polar residue, particularly S; and X284 is anon-polar residue, particularly G; X297 is a polar residue, particularlyS; and X321 is a constrained residue, particularly P. In someembodiments, the transaminase polypeptides can have additionally 1-2,1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16,1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35, 1-40, 1-45, 1-50, 1-55, or1-60 residue differences at other residue positions. In someembodiments, the number of differences can be 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35, 40, 45, 50, 55, or60 residue differences at the other residue positions. In someembodiments, the engineered transaminase polypeptide can comprise anamino acid sequence that is at least about 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to areference sequence based on SEQ ID NO:2 having the features describedfor preceding specified residue positions (e.g., SEQ ID NO:84, 86, 88,96, 98, or 100), with the proviso that the engineered transaminasepolypeptide comprises an amino acid sequence that includes at least thefeatures described for the specified residue positions. In someembodiments, the engineered transaminase polypeptide can comprise anamino acid sequence that is at least about 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to areference sequence of SEQ ID NO:84, 86, 88, 96, 98, or 100.

In some embodiments, the engineered transaminase comprises an amino acidsequence that includes the following features: X8 is a constrainedresidue, particularly is P; X60 is an aromatic residue, particularly F;X61 is an aromatic residue, particularly Y; X62 is an aromatic or polarresidue, particularly T, Y or F; X65 is an aliphatic residue,particularly A; X69 is C or a non-polar, aliphatic or polar residue,particularly G, C, T, A, or S; X81 is a non-polar residue, particularlyG; X94 is an aliphatic residue, particularly I or L; X96 is an aliphaticresidue, particularly L; X122 is a constrained, non-polar or aliphaticresidue, particularly M, I, L, V, or H; X136 is an aromatic residue,particularly Y or F; X150 is aromatic, constrained or polar residue,particularly F, H, or S; X169 is an aliphatic residue, particularly L;X199 is an aliphatic or aromatic residue, particularly W or I; X209 isan aliphatic residue, particularly L; X215 is C; X217 is a polarresidue, particularly N; X223 is a constrained residue, particularly P;X269 is a constrained residue, particularly P; X273 is an aromaticresidue, particularly Y; X282 is a polar residue, particularly S; andX284 is a non-polar residue, particularly G; X297 is a polar residue,particularly S; and X321 is a constrained residue, particularly P. Insome embodiments, the transaminase polypeptides can have additionally1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15,1-16, 1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35, 1-40, 1-45, 1-50, 1-55,or 1-60 residue differences at other residue positions. In someembodiments, the number of differences can be 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35, 40, 45, 50, 55, or60 residue differences at the other residue positions. In someembodiments, the engineered transaminase polypeptide can comprise anamino acid sequence that is at least about 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to areference sequence based on SEQ ID NO:2 having the features describedfor preceding specified residue positions (e.g., SEQ ID NO:90), with theproviso that the engineered transaminase polypeptide comprises an aminoacid sequence that includes at least the features described for thespecified residue positions. In some embodiments, the engineeredtransaminase polypeptide can comprise an amino acid sequence that is atleast about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% identical to a reference sequence of SEQ ID NO:90.

In some embodiments, the engineered transaminase comprises an amino acidsequence that includes the following features: X8 is a constrainedresidue, particularly is P; X60 is an aromatic residue, particularly F;X61 is an aromatic residue, particularly Y; X62 is an aromatic or polarresidue, particularly T, Y or F; X65 is an aliphatic residue,particularly A; X69 is C or a non-polar, aliphatic or polar residue,particularly G, C, T, A, or S; X81 is a non-polar residue, particularlyG; X94 is an aliphatic residue, particularly I or L; X122 is aconstrained, non-polar or aliphatic residue, particularly M, I, L, V, orH; X124 is a polar or constrained residue, particularly T, H or N; X136is an aromatic residue, particularly Y or F; X150 is aromatic,constrained or polar residue, particularly F, H, or S; X152 is C or anon-polar, aliphatic, or polar residue, particularly G, I, L, S or C;X169 is an aliphatic residue, particularly L; X199 is an aliphatic oraromatic residue, particularly W or I; X209 is an aliphatic residue,particularly L; X215 is a C; X217 is a polar residue, particularly N;X223 is a constrained residue, particularly P; X269 is a constrainedresidue, particularly P; X273 is an aromatic residue, particularly Y;X282 is a polar residue, particularly S; and X284 is a non-polarresidue, particularly G; X297 is a polar residue, particularly S; andX321 is a constrained residue, particularly P. In some embodiments, thetransaminase polypeptides can have additionally 1-2, 1-3, 1-4, 1-5, 1-6,1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22,1-24, 1-26, 1-30, 1-35, 1-40, 1-45, 1-50, 1-55, or 1-60 residuedifferences at other residue positions. In some embodiments, the numberof differences can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16,18, 20, 22, 24, 26, 30, 35, 40, 45, 50, 55, or 60 residue differences atthe other residue positions. In some embodiments, the engineeredtransaminase polypeptide can comprise an amino acid sequence that is atleast about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% identical to a reference sequence based on SEQ IDNO:2 having the features described for preceding specified residuepositions (e.g., SEQ ID NO:92), with the proviso that the engineeredtransaminase polypeptide comprises an amino acid sequence that includesat least the features described for the specified residue positions. Insome embodiments, the engineered transaminase polypeptide can comprisean amino acid sequence that is at least about 80%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to areference sequence of SEQ ID NO:92.

In some embodiments, the engineered transaminase comprises an amino acidsequence that includes the following features: X8 is a constrainedresidue, particularly is P; X60 is an aromatic residue, particularly F;X61 is an aromatic residue, particularly Y; X62 is an aromatic or polarresidue, particularly T, Y or F; X65 is an aliphatic residue,particularly A; X69 is C or a non-polar, aliphatic or polar residue,particularly G, C, T, A, or S; X81 is a non-polar residue, particularlyG; X94 is an aliphatic residue, particularly I or L; X96 is an aliphaticresidue, particularly L; X122 is a constrained, non-polar or aliphaticresidue, particularly M, I, L, V, or H; X124 is a polar or constrainedresidue, particularly T, H or N; X136 is an aromatic residue,particularly Y or F; X150 is aromatic, constrained or polar residue,particularly F, H, or S; X152 is C or a non-polar, aliphatic, or polarresidue, particularly G, I, L, S or C; X169 is an aliphatic residue,particularly L; X199 is an aliphatic or aromatic residue, particularly Wor I; X209 is an aliphatic residue, particularly L; X215 is a C; X217 isa polar residue, particularly N; X223 is a constrained residue,particularly P; X269 is a constrained residue, particularly P; X273 isan aromatic residue, particularly Y; X282 is a polar residue,particularly S; and X284 is a non-polar residue, particularly G; X297 isa polar residue, particularly S; and X321 is a constrained residue,particularly P. In some embodiments, the transaminase polypeptides canhave additionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11,1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35, 1-40,1-45, 1-50, 1-55, or 1-60 residue differences at other residuepositions. In some embodiments, the number of differences can be 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35,40, 45, 50, 55, or 60 residue differences at the other residuepositions. In some embodiments, the engineered transaminase polypeptidecan comprise an amino acid sequence that is at least about 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical to a reference sequence based on SEQ ID NO:2 having thefeatures described for preceding specified residue positions (e.g., SEQID NO:94), with the proviso that the engineered transaminase polypeptidecomprises an amino acid sequence that includes at least the featuresdescribed for the specified residue positions. In some embodiments, theengineered transaminase polypeptide can comprise an amino acid sequencethat is at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% identical to a reference sequence of SEQ IDNO:94.

In some embodiments, the engineered transaminase comprises an amino acidsequence that includes the following features: X8 is a constrainedresidue, particularly is P; X60 is an aromatic residue, particularly F;X61 is an aromatic residue, particularly Y; X62 is an aromatic or polarresidue, particularly T, Y or F; X65 is an aliphatic residue,particularly A; X69 is C or a non-polar, aliphatic or polar residue,particularly G, C, T, A, or S; X81 is a non-polar residue, particularlyG; X94 is an aliphatic residue, particularly I or L; X96 is an aliphaticresidue, particularly L; X122 is a constrained, non-polar or aliphaticresidue, particularly M, I, L, V, or H; X124 is a polar or constrainedresidue, particularly T, H or N; X136 is an aromatic residue,particularly Y or F; X169 is an aliphatic residue, particularly L; X199is an aliphatic or aromatic residue, particularly W or I; X209 is analiphatic residue, particularly L; X215 is C; X217 is a polar residue,particularly N; X223 is a constrained residue, particularly P; X269 is aconstrained residue, particularly P; X273 is an aromatic residue,particularly Y; X282 is a polar residue, particularly S; and X284 is anon-polar residue, particularly G; X297 is a polar residue, particularlyS; X321 is a constrained residue, particularly P; and X329 is aconstrained or aromatic residue, particularly H. In some embodiments,the transaminase polypeptides can have additionally 1-2, 1-3, 1-4, 1-5,1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20,1-22, 1-24, 1-26, 1-30, 1-35, 1-40, 1-45, 1-50, 1-55, or 1-60 residuedifferences at other residue positions. In some embodiments, the numberof differences can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16,18, 20, 22, 24, 26, 30, 35, 40, 45, 50, 55, or 60 residue differences atthe other residue positions. In some embodiments, the engineeredtransaminase polypeptide can comprise an amino acid sequence that is atleast about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% identical to a reference sequence based on SEQ IDNO:2 having the features described for preceding specified residuepositions (e.g., SEQ ID NO:102), with the proviso that the engineeredtransaminase polypeptide comprises an amino acid sequence that includesat least the features described for the specified residue positions. Insome embodiments, the engineered transaminase polypeptide can comprisean amino acid sequence that is at least about 80%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to areference sequence of SEQ ID NO:102.

In some embodiments, the engineered transaminase comprises an amino acidsequence that includes the following features: X8 is a constrainedresidue, particularly is P; X60 is an aromatic residue, particularly F;X61 is an aromatic residue, particularly Y; X62 is an aromatic or polarresidue, particularly T, Y or F; X65 is an aliphatic residue,particularly A; X69 is C or a non-polar, aliphatic or polar residue,particularly G, C, T, A, or S; X81 is a non-polar residue, particularlyG; X94 is an aliphatic residue, particularly I or L; X96 is an aliphaticresidue, particularly L; X122 is a constrained, non-polar or aliphaticresidue, particularly M, I, L, V, or H; X124 is a polar or constrainedresidue, particularly T, H or N; X136 is an aromatic residue,particularly Y or F; X150 is aromatic, constrained or polar residue,particularly S; X152 is cysteine (C), non-polar, aliphatic, or polarresidue, particularly G, I, L, S or C; X169 is an aliphatic residue,particularly L; X199 is an aliphatic or aromatic residue, particularly Wor I; X209 is an aliphatic residue, particularly L; X215 is C; X217 is apolar residue, particularly N; X223 is a constrained residue,particularly P; X269 is a constrained residue, particularly P; X273 isan aromatic residue, particularly Y; X282 is a polar residue,particularly S; X284 is a non-polar residue, particularly G; X297 is apolar residue, particularly S; and X321 is a constrained residue,particularly P. In some embodiments, the transaminase polypeptides canhave additionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11,1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35, 1-40,1-45, 1-50, 1-55, or 1-60 residue differences at other residuepositions. In some embodiments, the number of differences can be 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35,40, 45, 50, 55, or 60 residue differences at the other residuepositions. In some embodiments, the engineered transaminase polypeptidecan comprise an amino acid sequence that is at least about 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical to a reference sequence based on SEQ ID NO:2 having thefeatures described for preceding specified residue positions (e.g., SEQID NO:110), with the proviso that the engineered transaminasepolypeptide comprises an amino acid sequence that includes at least thefeatures described for the specified residue positions. In someembodiments, the engineered transaminase polypeptide can comprise anamino acid sequence that is at least about 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to areference sequence of SEQ ID NO:110.

In some embodiments, the engineered transaminase comprises an amino acidsequence that includes the following features: X8 is a constrainedresidue, particularly is P; X49 is a polar residue, particularly T; X60is an aromatic residue, particularly F; X61 is an aromatic residue,particularly Y; X62 is an aromatic or polar residue, particularly T, Yor F; X65 is an aliphatic residue, particularly A; X69 is C or anon-polar, aliphatic or polar residue, particularly G, C, T, A, or S;X81 is a non-polar residue, particularly G; X94 is an aliphatic residue,particularly I or L; X96 is an aliphatic residue, particularly L; X117is a non-polar residue, particularly G; X122 is a constrained, non-polaror aliphatic residue, particularly M, I, L, V, or H; X124 is a polar orconstrained residue, particularly T, H or N; X126 is a polar residue,particularly T; X136 is an aromatic residue, particularly Y or F; X150is aromatic, constrained or polar residue, particularly S; X152 iscysteine (C), non-polar, aliphatic, or polar residue, particularly G, I,L, S or C; X169 is an aliphatic residue, particularly L; X199 is analiphatic or aromatic residue, particularly W or I; X209 is an aliphaticresidue, particularly L; X215 is C; X217 is a polar residue,particularly N; X223 is a constrained residue, particularly P; X269 is aconstrained residue, particularly P; X273 is an aromatic residue,particularly Y; X282 is a polar residue, particularly S; X284 is anon-polar residue, particularly G; X297 is a polar residue, particularlyS; X302 is an aliphatic residue, particularly A; and X321 is aconstrained residue, particularly P. In some embodiments, thetransaminase polypeptides can have additionally 1-2, 1-3, 1-4, 1-5, 1-6,1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22,1-24, 1-26, 1-30, 1-35, 1-40, 1-45, 1-50, 1-55, or 1-60 residuedifferences at other residue positions. In some embodiments, the numberof differences can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16,18, 20, 22, 24, 26, 30, 35, 40, 45, 50, 55, or 60 residue differences atthe other residue positions. In some embodiments, the engineeredtransaminase polypeptide can comprise an amino acid sequence that is atleast about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% identical to a reference sequence based on SEQ IDNO:2 having the features described for preceding specified residuepositions (e.g., SEQ ID NO:166), with the proviso that the engineeredtransaminase polypeptide comprises an amino acid sequence that includesat least the features described for the specified residue positions. Insome embodiments, the engineered transaminase polypeptide can comprisean amino acid sequence that is at least about 80%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to areference sequence of SEQ ID NO:166.

Table 2 below provides exemplary engineered transaminase polypeptides,with each row listing two SEQ ID NOs, the odd number referring to thenucleotide sequence encoding the amino acid sequence provided by theeven number. The residue differences are based on comparison toreference sequence of SEQ ID NO:2, a transaminase derived fromArthrobacter sp KNK168 and differs from the naturally occurring enzymein having a substitution of isoleucine (I) at residue position X306 withvaline (V). In the Activity column, the levels of increasing activity(i.e., “+” “++” “+++” etc.) were defined as follows: “+” indicates atleast equal to but not greater than 2 times the activity of SEQ ID NO:4(assay conditions: 2 g/L ketoamide substrate, 0.5 M isopropylamine, 22°C., pH 7.5, 5% DMSO, 100 μM PLP); “++” indicates about 50-to-100 timesgreater than the activity of SEQ ID NO:4 (assay conditions: 2 g/Lketoamide substrate, 0.5 M isopropylamine, 22° C., pH 7.5, 5% MeOH, 100μM PLP); “+++” indicates about 1.1 to about 5 times greater than theactivity of SEQ ID NO:22 (assay conditions: 5-10 g/L ketoamidesubstrate, 0.5-1 M isopropylamine, 22-30° C., pH 7.5, 5% MeOH, 100 μMPLP); “++++” indicates about 1.1 to 5 times greater than the activity ofSEQ ID NO:48 (assay conditions: 10-40 g/L ketoamide substrate, 1 Misopropylamine, 30-45° C., pH 8.5, 10% MeOH, 100 μM PLP); “+++++”indicates about 1.1 to 5 times or greater than the activity of SEQ IDNO:58 (assay conditions: 40-100 g/L ketoamide substrate, 1 Misopropylamine, 45° C., pH 8.5, 10% MeOH-25% DMSO, 250 μM PLP); “++++++”indicates about 1.1 to 5 times or greater than the activity of SEQ IDNO:104 (assay conditions: 40-100 g/L ketoamide substrate, 1 Misopropylamine, 45° C., pH 8.5, 50% DMSO, 1000 μM PLP). Exemplary assayconditions for measuring activity using methanol and DMSO are describedin Examples 6-11.

TABLE 2 No. SEQ ID Residue NO Residue differences relative to SEQ ID NO:2 Differences Activity 1/2 — — — 3/4 V69G; F122V; S223P; A284G 4 + 5/6V65A; V69G; F122I; S223P 4 + 7/8 F122L; S223P; A284G 3 +  9/10 F122I;S223P; A284G 3 + 11/12 F122L; S174A; S223P; A284G 4 + 13/14 Y26H; V65A;V69G; F122I; S223P; A284G 6 + 15/16 Y26H; H62T; V65A; V69G; F122I;T178S; V199W; S223P; 11 ++ F225Y; T282S; A284G 17/18 Y26H; H62F; V65A;V69G; F122V; G136Y; V199I; A209L; 12 ++ S223P; F225Y; T282S; A284G 19/20Y26H; H62T; V65A; V69G; F122V; G136Y; E137T; V199I; 12 ++ A209L; S223P;T282S; A284G 21/22 Y26H; H62T; V65A; V69G; F122I; G136Y; E137I; V199I;A209L; 12 ++ S223P; T282S; A284G 23/24 Y26H; H62T; V65A; V69G; F122I;G136Y; E137T; V199I; A209L; 13 ++ S223P; F225Y; T282S; A284G 25/26 Y26H;V65A; V69G; F122V; G136Y; E137I; S174A; V199I; 12 ++ A209L; S223P;I230V; A284G 27/28 Y26H; H62T; V65A; V69G; F122H; G136Y; E137I; V199I;12 +++ A209L; S223P; T282S; A284G 29/30 Y26H; H62T; V65A; V69T; F122I;G136Y; E137I; V199I; A209L; 12 +++ S223P; T282S; A284G 31/32 Y26H; H62T;V65A; V69C; F122I; G136Y; E137I; V199I; A209L; 12 +++ S223P; T282S;A284G 33/34 Y26H; H62T; V65A; V69A; F122I; G136Y; E137I; V199I; A209L;12 +++ S223P; T282S; A284G 35/36 Y26H; L61Y; H62T; V65A; V69G; F122I;G136Y; E137I; V199I; 13 +++ A209L; S223P; T282S; A284G 37/38 Y26H; H62Y;V65A; V69G; F122I; G136Y; E137I; V199I; A209L; 12 +++ S223P; T282S;A284G 39/40 Y26H; H62T; V65A; V69G; F122I; G136F; E137I; V199I; A209L;12 +++ S223P; T282S; A284G 41/42 S4Y; Y26H; H62T; V65A; V69G; M94I;F122I; G136Y; E137T; 15 +++ V199I; A209L; G215C; S223P; T282S; A284G43/44 H62T; V65A; V69G; M94I; F122I; G136Y; E137I; V199I; A209L; 13 +++G215C; S223P; T282S; A284G 45/46 H62T; V65A; V69G; M94I; F122I; G136Y;E137T; V199I; A209L; 13 +++ G215C; S223P; T282S; A284G 47/48 H62T; V65A;V69C; M94I; F122I; G136Y; E137T; V199I; A209L; 13 +++ G215C; S223P;T282S; A284G 49/50 S8P; H62T; V65A; V69C; M94I; F122I; G136Y; E137T;V199I; 14 ++++ A209L; G215C; S223P; T282S; A284G 51/52 L61Y; H62T; V65A;V69S; M94I; F122I; G136F; E137T; V152I; 15 ++++ V199I; A209L; G215C;S223P; T282S; A284G 53/54 L61Y; H62T; V65A; V69C; M94I; F122V; G136F;E137T; V199I; 14 ++++ A209L; G215C; S223P; T282S; A284G 55/56 L61Y;H62T; V65A; V69T; M94I; F122V; G136F; E137T; V199I; 14 ++++ A209L;G215C; S223P; T282S; A284G 57/58 L61Y; H62T; V65A; V69T; M94I; F122I;G136F; V199I; A209L; 13 ++++ G215C; S223P; T282S; A284G 59/60 L61Y;H62T; V65A; V69T; M94I; F122H; G136F; V199I; A209L; 13 ++++ G215C;S223P; T282S; A284G 61/62 L61Y; H62T; V65A; V69C; M94I; F122I; G136Y;E137T; F160L; 17 ++++ A169L; V199I; A209L; G215C; S223P; L269P; T282S;A284G 63/64 L61Y; H62T; V65A; V69C; M94L; F122I; G136Y; E137T; A169L; 16++++ V199I; A209L; G215C; S223P; T282S; A284G; V306L 65/66 L61Y; H62T;V65A; V69C; M94I; Q102L; F122I; G136F; Y150F; 16 ++++ V152I; V199I;A209L; G215C; S223P; T282S; A284G 67/68 S8P; P48A; L61Y; H62T; V65A;V69T; D81G; M94I; I96L; 24 +++++ Q102K; F122I; G136F; I163V; V199I;A209L; L211I; G215C; G217N; S223P; L252F; L273Y; T282S; A284G; S321P69/70 S8P; P48A; L61Y; H62T; V65A; V69T; D81G; M94I; F122I; 21 +++++G136F; A169L; V199I; A209L; G215C; G217N; S223P; L269P; T282S; A284G;P297S; S321P 71/72 S8P; L61Y; H62T; V65A; V69T; M94I; F122I; G136F;V199I; 14 +++++ A209L; G215C; S223P; T282S; A284G 73/74 S8P; L61Y; H62T;V65A; V69T; D81G; M94L; I96L; F122I; 20 +++++ G136F; T178S; V199I;A209L; G215C; S223P; L269P; T282S; A284G; P297S; S321P 75/76 S8P; Y60F;L61Y; H62T; V65A; V69T; D81G; M94I; I96L; F122I; 25 +++++ G136F; V152L;T178S; V199I; A209L; G215C; G217N; S223P; L252F; L269P; L273Y; T282S;A284G; P297S; S321P 77/78 S8P; Y60F; L61Y; H62T; V65A; V69T; D81G; M94L;I96L; F122I; 24 +++++ G136F; A169L; T178S; V199I; A209L; G215C; G217N;S223P; L269P; T282S; A284G; S292T; P297S; S321P 79/80 S8P; Y60F; L61Y;H62T; V65A; V69T; D81G; M94I; I96L; F122I; 23 +++++ G136F; A169L; V199I;A209L; G215C; G217N; S223P; L269P; L273Y; T282S; A284G; P297S; S321P81/82 S8P; Y60F; L61Y; H62T; V65A; V69T; D81G; M94L; I96L; F122I; 23+++++ G136F; A169L; T178S; V199I; A209L; G215C; S223P; L269P; L273Y;T282S; A284G; P297S; S321P 83/84 S8P; Y60F; L61Y; H62T; V65A; V69T;D81G; M94I; I96L; F122I; 24 +++++ S124T; G136F; A169L; V199I; A209L;G215C; G217N; S223P; L269P; L273Y; T282S; A284G; P297S; S321P 85/86 S8P;Y60F; L61Y; H62T; V65A; V69T; D81G; M94I; I96L; F122I; 24 +++++ S124H;G136F; A169L; V199I; A209L; G215C; G217N; S223P; L269P; L273Y; T282S;A284G; P297S; S321P 87/88 S8P; Y60F; L61Y; H62T; V65A; V69T; D81G; M94I;I96L; F122I; 24 +++++ S124N; G136F; A169L; V199I; A209L; G215C; G217N;S223P; L269P; L273Y; T282S; A284G; P297S; S321P 89/90 S8P; Y60F; L61Y;H62T; V65A; V69T; D81G; M94I; I96L; F122I; 24 +++++ G136F; Y150H; A169L;V199I; A209L; G215C; G217N; S223P; L269P; L273Y; T282S; A284G; P297S;S321P 91/92 S8P; Y60F; L61Y; H62T; V65A; V69T; D81G; M94I; I96L; 26+++++ F122M; S124H; G136F; Y150H; V152S; A169L; V199I; A209L; G215C;G217N; S223P; L269P; L273Y; T282S; A284G; P297S; S321P 93/94 S8P; Y60F;L61Y; H62T; V65A; V69T; D81G; M94I; I96L; F122I; 26 +++++ S124T; G136F;Y150S; V152C; A169L; V199I; A209L; G215C; G217N; S223P; L269P; L273Y;T282S; A284G; P297S; S321P 95/96 S8P; Y60F; L61Y; H62T; V65A; V69T;D81G; M94I; I96L; 24 +++++ F122M; S124N; G136F; A169L; V199I; A209L;G215C; G217N; S223P; L269P; L273Y; T282S; A284G; P297S; S321P 97/98 S8P;Y60F; L61Y; H62T; V65A; V69T; D81G; M94I; I96L; F122I; 24 +++++ S124H;G136F; A169L; V199I; A209L; G215C; G217N; S223P; L269P; L273Y; T282S;A284G; P297S; S321P  99/100 S8P; Y60F; L61Y; H62T; V65A; V69T; D81G;M94I; I96L; 24 +++++ F122M; S124N; G136F; A169L; V199I; A209L; G215C;G217N; S223P; L269P; L273Y; T282S; A284G; P297S; S321P 101/102 S8P;Y60F; L61Y; H62T; V65A; V69T; D81G; M94I; I96L; 25 +++++ F122M; S124N;G136F; A169L; V199I; A209L; G215C; G217N; S223P; L269P; L273Y; T282S;A284G; P297S; S321P; Q329H 103/104 S8P; Y60F; L61Y; H62T; V65A; V69T;D81G; M94I; I96L; 26 +++++ F122M; S124T; G136F; Y150S; V152C; A169L;V199I; A209L; G215C; G217N; S223P; L269P; L273Y; T282S; A284G; P297S;S321P 105/106 S8P; Y60F; L61Y; H62T; V65A; V69T; D81G; M94I; I96L; 29+++++ F122M; S124T; S126T; G136F; R138K; Y150S; V152G; Q155M; A169L;V199I; A209L; G215C; G217N; S223P; L269P; L273Y; T282S; A284G; P297S;S321P 107/108 S8P; Y60F; L61Y; H62T; V65A; V69T; D81G; M94I; I96L; 28+++++ F122M; S124T; G136F; R138P; Q146R; Y150S; V152S; A169L; V199I;A209L; G215C; G217N; S223P; L269P; L273Y; T282S; A284G; P297S; S321P109/110 S8P; Y60F; L61Y; H62T; V65A; V69T; D81G; M94I; I96L; 27 ++++++F122M; S124T; S126T; G136F; Y150S; V152C; A169L; V199I; A209L; G215C;G217N; S223P; L269P; L273Y; T282S; A284G; P297S; S321P 111/112 S8P;Y60F; L61Y; H62T; V65A; V69T; D81G; M94I; I96L; 27 ++++++ F122M; S124T;G136F; Y150S; V152C; I163H; A169L; V199I; A209L; G215C; G217N; S223P;L269P; L273Y; T282S; A284G; P297S; S321P 113/114 S8P; Y60F; L61Y; H62T;V65A; V69T; D81G; M94I; I96L; 27 ++++++ F122M; S124T; G136F; Y148A;Y150S; V152C; A169L; V199I; A209L; G215C; G217N; S223P; L269P; L273Y;T282S; A284G; P297S; S321P 115/116 S8P; Y60F; L61Y; H62T; V65A; V69T;D81G; M94I; I96L; 27 ++++++ F122M; S124T; G136F; Y150S; V152C; W156Q;A169L; V199I; A209L; G215C; G217N; S223P; L269P; L273Y; T282S; A284G;P297S; S321P 117/118 S8P; Y60F; L61Y; H62T; V65A; V69T; D81G; M94I;I96L; 27 ++++++ F122M; S124T; G136F; Y150S; V152C; R164V; A169L; V199I;A209L; G215C; G217N; S223P; L269P; L273Y; T282S; A284G; P297S; S321P119/120 S8P; Y60F; L61Y; H62T; V65A; V69T; D81G; M94I; I96L; 27 ++++++F122M; S124T; G136F; Y148F; Y150S; V152C; A169L; V199I; A209L; G215C;G217N; S223P; L269P; L273Y; T282S; A284G; P297S; S321P 121/122 S8P;Y60F; L61Y; H62T; V65A; V69T; D81G; M94I; I96L; 27 ++++++ E120Y; F122M;S124T; G136F; Y150S; V152C; A169L; V199I; A209L; G215C; G217N; S223P;L269P; L273Y; T282S; A284G; P297S; S321P 123/124 S8P; Y60F; L61Y; H62T;V65A; V69T; D81G; M94I; I96L; 27 ++++++ F122M; S124T; G136F; Y150S;V152C; Q155V; A169L; V199I; A209L; G215C; G217N; S223P; L269P; L273Y;T282S; A284G; P297S; S321P 125/126 S8P; Y60F; L61Y; H62T; V65A; V69T;D81G; M94I; I96L; 27 ++++++ F122M; S124T; G136F; Y150S; V152C; R164P;A169L; V199I; A209L; G215C; G217N; S223P; L269P; L273Y; T282S; A284G;P297S; S321P 127/128 S8P; Y60F; L61Y; H62T; V65A; V69T; D81G; M94I;I96L; 27 ++++++ F122M; S124T; G136F; Y150S; V152C; Q155T; A169L; V199I;A209L; G215C; G217N; S223P; L269P; L273Y; T282S; A284G; P297S; S321P129/130 S8P; E50L; Y60F; L61Y; H62T; V65A; V69T; D81G; M94I; I96L; 27++++++ F122M; S124T; G136F; Y150S; V152C; A169L; V199I; A209L; G215C;G217N; S223P; L269P; L273Y; T282S; A284G; P297S; S321P 131/132 S8P;L18I; Y60F; L61Y; H62T; V65A; V69T; D81G; M94I; I96L; 27 ++++++ F122M;S124T; G136F; Y150S; V152C; A169L; V199I; A209L; G215C; G217N; S223P;L269P; L273Y; T282S; A284G; P297S; S321P 133/134 S8P; D25Q; Y60F; L61Y;H62T; V65A; V69T; D81G; M94I; I96L; 27 ++++++ F122M; S124T; G136F;Y150S; V152C; A169L; V199I; A209L; G215C; G217N; S223P; L269P; L273Y;T282S; A284G; P297S; S321P 135/136 S8P; E42G; Y60F; L61Y; H62T; V65A;V69T; D81G; M94I; I96L; 27 ++++++ F122M; S124T; G136F; Y150S; V152C;A169L; V199I; A209L; G215C; G217N; S223P; L269P; L273Y; T282S; A284G;P297S; S321P 137/138 S8P; P48D; Y60F; L61Y; H62T; V65A; V69T; D81G;M94I; I96L; 27 ++++++ F122M; S124T; G136F; Y150S; V152C; A169L; V199I;A209L; G215C; G217N; S223P; L269P; L273Y; T282S; A284G; P297S; S321P139/140 S8P; P30Q; Y60F; L61Y; H62T; V65A; V69T; D81G; M94I; I96L; 27++++++ F122M; S124T; G136F; Y150S; V152C; A169L; V199I; A209L; G215C;G217N; S223P; L269P; L273Y; T282S; A284G; P297S; S321P 141/142 S8P;L28P; Y60F; L61Y; H62T; V65A; V69T; D81G; M94I; I96L; 27 ++++++ F122M;S124T; G136F; Y150S; V152C; A169L; V199I; A209L; G215C; G217N; S223P;L269P; L273Y; T282S; A284G; P297S; S321P 143/144 S8P; I41H; Y60F; L61Y;H62T; V65A; V69T; D81G; M94I; I96L; 27 ++++++ F122M; S124T; G136F;Y150S; V152C; A169L; V199I; A209L; G215C; G217N; S223P; L269P; L273Y;T282S; A284G; P297S; S321P 145/146 S8P; P30M; Y60F; L61Y; H62T; V65A;V69T; D81G; M94I; I96L; 27 ++++++ F122M; S124T; G136F; Y150S; V152C;A169L; V199I; A209L; G215C; G217N; S223P; L269P; L273Y; T282S; A284G;P297S; S321P 147/148 S8P; S54H; Y60F; L61Y; H62T; V65A; V69T; D81G;M94I; I96L; 27 ++++++ F122M; S124T; G136F; Y150S; V152C; A169L; V199I;A209L; G215C; G217N; S223P; L269P; L273Y; T282S; A284G; P297S; S321P149/150 S8P; L18C; I55V; Y60F; L61Y; H62T; V65A; V69T; D81G; M94I; 28++++++ I96L; F122M; S124T; G136F; Y150S; V152C; A169L; V199I; A209L;G215C; G217N; S223P; L269P; L273Y; T282S; A284G; P297S; S321P 151/152S8P; P48G; Y60F; L61Y; H62T; V65A; V69T; D81G; M94I; I96L; 27 ++++++F122M; S124T; G136F; Y150S; V152C; A169L; V199I; A209L; G215C; G217N;S223P; L269P; L273Y; T282S; A284G; P297S; S321P 153/154 S8P; P48V; Y60F;L61Y; H62T; V65A; V69T; D81G; M94I; I96L; 27 ++++++ F122M; S124T; G136F;Y150S; V152C; A169L; V199I; A209L; G215C; G217N; S223P; L269P; L273Y;T282S; A284G; P297S; S321P 155/156 S8P; 14IS; Y60F; L61Y; H62T; V65A;V69T; D81G; M94I; I96L; 27 ++++++ F122M; S124T; G136F; Y150S; V152C;A169L; V199I; A209L; G215C; G217N; S223P; L269P; L273Y; T282S; A284G;P297S; S321P 157/158 S8P; E27T; Y60F; L61Y; H62T; V65A; V69T; D81G;M94I; I96L; 27 ++++++ F122M; S124T; G136F; Y150S; V152C; A169L; V199I;A209L; G215C; G217N; S223P; L269P; L273Y; T282S; A284G; P297S; S321P159/160 S8P; S54P; Y60F; L61Y; H62T; V65A; V69T; D81G; M94I; I96L; 27++++++ F122M; S124T; G136F; Y150S; V152C; A169L; V199I; A209L; G215C;G217N; S223P; L269P; L273Y; T282S; A284G; P297S; S321P 161/162 S8P;P48Q; Y60F; L61Y; H62T; V65A; V69T; D81G; M94I; I96L; 27 ++++++ F122M;S124T; G136F; Y150S; V152C; A169L; V199I; A209L; G215C; G217N; S223P;L269P; L273Y; T282S; A284G; P297S; S321P 163/164 A5K; S8P; Y60F; L61Y;H62T; V65A; V69T; D81G; M94I; I96L; 28 ++++++ F122M; S124T; S126T;G136F; Y150S; V152C; A169L; V199I; A209L; G215C; G217N; S223P; L269P;L273Y; T282S; A284G; P297S; S321P 165/166 S8P; S49T; Y60F; L61Y; H62T;V65A; V69T; D81G; M94I; I96L; 30 ++++++ E117G; F122M; S124T; S126TG136F; Y150S; V152S; A169L; V199I; A209L; G215C; G217N; S223P; L269P;L273Y; T282S; A284G; P297S; V302A; S321P 167/168 S8P; S54P; Y60F; L61Y;H62T; V65A; V69T; D81G; M94I; I96L; 29 ++++++ F122M; S124T; S126T;G136F; Y150S; V152S; A169L; V199I; D204A; A209L; G215C; G217N; S223P;L269P; L273Y; T282S; A284G; P297S; S321P

As noted above, in some embodiments, the transaminase polypeptide cancomprise an amino acid sequence that is at least about 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moreidentical to a reference sequence of SEQ ID NO: 6, 8, 10, 12, 14, 16,18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52,54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88,90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118,120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146,148, 150, 152, 154, 156, 158, 160, 162, 164, 166 or 168. In someembodiments, the transaminase polypeptides can have 1-2, 1-3, 1-4, 1-5,1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20,1-22, 1-24, 1-26, 1-30, 1-35, 1-40, 1-45, 1-50, 1-55, or 1-60 residuedifferences as compared to the naturally occurring transaminaserepresented by SEQ ID NO:2. In some embodiments, the number of residuedifferences can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16,18, 20, 22, 24, 26, 30, 35, 40, 45, 50, 55, or 60 differences ascompared to SEQ ID NO:2.

In some embodiments, the transaminase polypeptide comprises an aminoacid sequence that is at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to areference sequence of SEQ ID NO: 58, 72, 74, 80, 86, 96, 98, 100, or102. In some embodiments, the transaminase polypeptides can have 1-2,1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16,1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35, 1-40, 1-45, 1-50, 1-55, or1-60 residue differences as compared to the naturally occurringtransaminase represented by SEQ ID NO:2. In some embodiments, the numberof residue differences can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14,15, 16, 18, 20, 22, 24, 26, 30, 35, 40, 45, 50, 55, or 60 differences ascompared to SEQ ID NO:2.

In some embodiments, the transaminase polypeptide comprises an aminoacid sequence that is at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a referencesequence based on SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60,62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96,98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124,126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152,154, 156, 158, 160, 162, 164, 166 or 168, with the proviso that thetransaminase amino acid sequence comprises any one of the set of residuedifferences contained in any one of the polypeptide sequences listed inTable 2 as compared to SEQ ID NO:2. In some embodiments, thetransaminase polypeptides can have additionally 1-2, 1-3, 1-4, 1-5, 1-6,1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22,1-24, 1-26, 1-30, 1-35, 1-40, 1-45, 1-50, 1-55, or 1-60 residuedifferences at other amino acid residue positions as compared to thereference sequence. In some embodiments, the number of differences canbe 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 18, 20, 22, 24,26, 30, 35, 40, 45, 50, 55, or 60 residue differences at other residuepositions. In some embodiments, the residue differences at other residuepositions comprise substitutions with conservative amino acid residues.

As noted above, in some embodiments, the transaminase polypeptide isalso capable of converting keto substrates to amino products with atleast 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%enantiomeric excess. Exemplary transaminase polypeptides with thespecified levels of enantioselectivity can comprise an amino acidsequence corresponding to SEQ ID NO: 58, 72, 74, 80, 86, 96, 98, 100,102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128,130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156,158, 160, 162, 164, 166 or 168.

In some embodiments, the engineered transaminase polypeptides cancomprise deletions of the engineered transaminase polypeptides describedherein. Thus, for each and every embodiment of the transaminasepolypeptides of the disclosure, the deletions can comprise one or moreamino acids, 2 or more amino acids, 3 or more amino acids, 4 or moreamino acids, 5 or more amino acids, 6 or more amino acids, 8 or moreamino acids, 10 or more amino acids, 15 or more amino acids, or 20 ormore amino acids, up to 10% of the total number of amino acids, up to10% of the total number of amino acids, up to 20% of the total number ofamino acids, or up to 30% of the total number of amino acids of thetransaminase polypeptides, as long as the functional activity of thetransaminase activity is maintained. In some embodiments, the deletionscan comprise, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12,1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35, 1-40, 1-45,1-50, 1-55, or 1-60 amino acid residues. In some embodiments, the numberof deletions can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16,18, 20, 22, 24, 26, 30, 35, 40, 45, 50, 55, or 60 amino acids. In someembodiments, the deletions can comprise deletions of 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 22, 24, 26, 28, or 30 aminoacid residues.

As described herein, the transaminase polypeptides of the disclosure canbe in the form of fusion polypeptides in which the transaminasepolypeptides are fused to other polypeptides, such as, by way of exampleand not limitation, antibody tags (e.g., myc epitope), purificationssequences (e.g., His tags for binding to metals), and cell localizationsignals (e.g., secretion signals). Thus, the transaminase polypeptidescan be used with or without fusions to other polypeptides.

The polypeptides described herein are not restricted to the geneticallyencoded amino acids. In addition to the genetically encoded amino acids,the polypeptides described herein may be comprised, either in whole orin part, of naturally-occurring and/or synthetic non-encoded aminoacids. Certain commonly encountered non-encoded amino acids of which thepolypeptides described herein may be comprised include, but are notlimited to: the D-stereoisomers of the genetically-encoded amino acids;2,3-diaminopropionic acid (Dpr); α-aminoisobutyric acid (Aib);ε-aminohexanoic acid (Aha); δ-aminovaleric acid (Ava); N-methylglycineor sarcosine (MeGly or Sar); ornithine (Orn); citrulline (Cit);t-butylalanine (Bua); t-butylglycine (Bug); N-methylisoleucine (MeIle);phenylglycine (Phg); cyclohexylalanine (Cha); norleucine (Nle);naphthylalanine (Nal); 2-chlorophenylalanine (Ocf);3-chlorophenylalanine (Mcf); 4-chlorophenylalanine (Pcf);2-fluorophenylalanine (Off); 3-fluorophenylalanine (Mff);4-fluorophenylalanine (Pff); 2-bromophenylalanine (Obf);3-bromophenylalanine (Mbf); 4-bromophenylalanine (Pbf);2-methylphenylalanine (Omf); 3-methylphenylalanine (Mmf);4-methylphenylalanine (Pmf); 2-nitrophenylalanine (Onf);3-nitrophenylalanine (Mnf); 4-nitrophenylalanine (Pnf);2-cyanophenylalanine (Ocf); 3-cyanophenylalanine (Mcf);4-cyanophenylalanine (Pcf); 2-trifluoromethylphenylalanine (Otf);3-trifluoromethylphenylalanine (Mtf); 4-trifluoromethylphenylalanine(Ptf); 4-aminophenylalanine (Paf); 4-iodophenylalanine (Pif);4-aminomethylphenylalanine (Pamf); 2,4-dichlorophenylalanine (Opef);3,4-dichlorophenylalanine (Mpcf); 2,4-difluorophenylalanine (Opff);3,4-difluorophenylalanine (Mpff); pyrid-2-ylalanine (2pAla);pyrid-3-ylalanine (3pAla); pyrid-4-ylalanine (4pAla); naphth-1-ylalanine(1nAla); naphth-2-ylalanine (2nAla); thiazolylalanine (taAla);benzothienylalanine (bAla); thienylalanine (tAla); furylalanine (fAla);homophenylalanine (hPhe); homotyrosine (hTyr); homotryptophan (hTrp);pentafluorophenylalanine (5ff); styrylkalanine (sAla); authrylalanine(aAla); 3,3-diphenylalanine (Dfa); 3-amino-5-phenypentanoic acid (Afp);penicillamine (Pen); 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid(Tic); β-2-thienylalanine (Thi); methionine sulfoxide (Mso);N(w)-nitroarginine (nArg); homolysine (hLys);phosphonomethylphenylalanine (pmPhe); phosphoserine (pSer);phosphothreonine (pThr); homoaspartic acid (hAsp); homoglutamic acid(hGlu); 1-aminocyclopent-(2 or 3)-ene-4 carboxylic acid; pipecolic acid(PA), azetidine-3-carboxylic acid (ACA);1-aminocyclopentane-3-carboxylic acid; allylglycine (aOly);propargylglycine (pgGly); homoalanine (hAla); norvaline (nVal);homoleucine (hLeu), homovaline (hVal); homoisoleucine (hIle);homoarginine (hArg); N-acetyl lysine (AcLys); 2,4-diaminobutyric acid(Dbu); 2,3-diaminobutyric acid (Dab); N-methylvaline (MeVal);homocysteine (hCys); homoserine (hSer); hydroxyproline (Hyp) andhomoproline (hPro). Additional non-encoded amino acids of which thepolypeptides described herein may be comprised will be apparent to thoseof skill in the art (see, e.g., the various amino acids provided inFasman, 1989, CRC Practical Handbook of Biochemistry and MolecularBiology, CRC Press, Boca Raton, Fla., at pp. 3-70 and the referencescited therein, all of which are incorporated by reference). These aminoacids may be in either the L- or D-configuration.

Those of skill in the art will recognize that amino acids or residuesbearing side chain protecting groups may also comprise the polypeptidesdescribed herein. Non-limiting examples of such protected amino acids,which in this case belong to the aromatic category, include (protectinggroups listed in parentheses), but are not limited to: Arg(tos),Cys(methylbenzyl), Cys (nitropyridinesulfenyl), Glu(δ-benzylester),Gln(xanthyl), Asn(N-δ-xanthyl), His(bom), His(benzyl), His(tos),Lys(fmoc), Lys(tos), Ser(O-benzyl), Thr (O-benzyl) and Tyr(O-benzyl).

Non-encoding amino acids that are conformationally constrained of whichthe polypeptides described herein may be composed include, but are notlimited to, N-methyl amino acids (L-configuration); 1-aminocyclopent-(2or 3)-ene-4-carboxylic acid; pipecolic acid; azetidine-3-carboxylicacid; homoproline (hero); and 1-aminocyclopentane-3-carboxylic acid.

As described above the various modifications introduced into thenaturally occurring polypeptide to generate an engineered transaminaseenzyme can be targeted to a specific property of the enzyme.

In another aspect, the present disclosure provides polynucleotidesencoding the improved transaminase polypeptides. The polynucleotides maybe operatively linked to one or more heterologous regulatory sequencesthat control gene expression to create a recombinant polynucleotidecapable of expressing the transaminase polypeptide. Expressionconstructs containing a heterologous polynucleotide encoding theengineered transaminase can be introduced into appropriate host cells toexpress the corresponding transaminase polypeptide.

Because of the knowledge of the codons corresponding to the variousamino acids, availability of a protein sequence provides a descriptionof all the polynucleotides capable of encoding the subject. Thedegeneracy of the genetic code, where the same amino acids are encodedby alternative or synonymous codons allows an extremely large number ofnucleic acids to be made, all of which encode the improved transaminasepolypeptides disclosed herein. Thus, having identified a particularamino acid sequence, those skilled in the art could make any number ofdifferent nucleic acids by simply modifying the sequence of one or morecodons in a way which does not change the amino acid sequence of theprotein. In this regard, the present disclosure specificallycontemplates each and every possible variation of polynucleotides thatcould be made by selecting combinations based on the possible codonchoices, and all such variations are to be considered specificallydisclosed for any polypeptide disclosed herein, including the amino acidsequences presented in Table 2.

In some embodiments, the polynucleotides can be selected and/orengineered to comprise codons that are preferably selected to fit thehost cell in which the protein is being produced. For example, preferredcodons used in bacteria are used to express the gene in bacteria;preferred codons used in yeast are used for expression in yeast; andpreferred codons used in mammals are used for expression in mammaliancells. Since not all codons need to be replaced to optimize the codonusage of the transaminases (e.g., because the natural sequence can havepreferred codons and because use of preferred codons may not be requiredfor all amino acid residues), codon optimized polynucleotides encodingthe transaminase polypeptides may contain preferred codons at about 40%,50%, 60%, 70%, 80%, or greater than 90% of codon positions of the fulllength coding region.

In some embodiments, the polynucleotide encodes a transaminasepolypeptide comprising an amino acid sequence that is at least 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore identical to the reference sequence of SEQ ID NO: 58, 72, 74, 80,86, 96, 98, 100, or 102, wherein the polypeptide is capable ofconverting the ketone substrate to the amine product with an activitythat is improved as compared to the activity of the naturally occurringtransaminase of Arthrobacter sp KNK168 or the transaminase of SEQ IDNO:2.

In some embodiments, the polynucleotide encodes a transaminasepolypeptide comprising an amino acid sequence that has at least about80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% or more sequence identity to the polypeptide comprising anamino acid sequence corresponding to SEQ ID NO: 4, 6, 8, 10, 12, 14, 16,18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52,54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88,90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118,120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146,148, 150, 152, 154, 156, 158, 160, 162, 164, 166 or 168. wherein thepolypeptide has one or more improved properties in converting the ketonesubstrate to the amine product in the presence of an amino group donor.In some embodiments, the encoded transaminase polypeptide has anactivity that is equal to or greater than the activity of thepolypeptide of SEQ ID NO:2.

In some embodiments, the polynucleotide encodes a transaminasepolypeptide comprising an amino acid sequence that is at least about80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to a reference sequence of SEQ ID NO: 4, 6, 8, 10,12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46,48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82,84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114,116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142,144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166 or 168.

In some embodiments, the polynucleotide encodes a transaminasepolypeptide comprising an amino acid sequence that is at least about80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to a reference sequence based on SEQ ID NO: 4, 6,8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42,44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78,80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110,112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138,140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166 or168, with the proviso that the improved transaminase amino acid sequencecomprises any one of the set of residue differences contained in any oneof the polypeptide sequences listed in Table 2 as compared to SEQ IDNO:2.

In some embodiments, the polynucleotides encoding the improvedtransaminase polypeptides are selected from SEQ ID NO: 3, 5, 7, 9, 11,13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47,49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83,85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115,117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143,145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, or 167.

In some embodiments, the polynucleotides are capable of hybridizingunder highly stringent conditions to a polynucleotide comprising SEQ IDNO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37,39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73,75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107,109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135,137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163,165, or 167, or a complement thereof, where the highly stringentlyhybridizing polynucleotides encode a transaminase polypeptide capable ofconverting the compounds of formula (II) to the amine products offormula (I) in presence of an amino group donor with an activity that isimproved as compared to the polypeptide of SEQ ID NO:2.

In some embodiments, the polynucleotides encode the polypeptidesdescribed herein but have about 80% or more sequence identity, about80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% or more sequence identity at the nucleotide level to areference polynucleotide encoding the engineered transaminase describedherein. In some embodiments, the reference polynucleotide is selectedfrom SEQ ID NO:3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31,33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67,69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101,103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129,131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157,159, 161, 163, 165, or 167.

An isolated polynucleotide encoding an improved transaminase polypeptidemay be manipulated in a variety of ways to provide for expression of thepolypeptide. In some embodiments, the polynucleotides encoding theengineered transaminase polypeptides can be provided as expressionvectors where one or more control sequences is present to regulate theexpression of the polynucleotides. Manipulation of the isolatedpolynucleotide prior to its insertion into a vector may be desirable ornecessary depending on the expression vector. The techniques formodifying polynucleotides and nucleic acid sequences utilizingrecombinant DNA methods are well known in the art. Guidance is providedin Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, 3rdEd., Cold Spring Harbor Laboratory Press; and Current Protocols inMolecular Biology, Ausubel. F. ed., Greene Pub. Associates, 1998,updates to 2006.

In some embodiments, the control sequences include among others,promoters, leader sequence, polyadenylation sequence, propeptidesequence, signal peptide sequence, and transcription terminator. Forbacterial host cells, suitable promoters for directing transcription ofthe nucleic acid constructs of the present disclosure, include thepromoters obtained from the E. coli lac operon, E. coli trp operon,bacteriophage λ, Streptomyces coelicolor agarase gene (dagA), Bacillussubtilis levansucrase gene (sacB), Bacillus licheniformis alpha-amylasegene (amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM),Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacilluslicheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylBgenes, and prokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978,Proc. Natl Acad. Sci. USA 75: 3727-3731), as well as the tac promoter(DeBoer et al., 1983, Proc. Natl Acad. Sci. USA 80: 21-25).

For filamentous fungal host cells, suitable promoters for directing thetranscription of the nucleic acid constructs of the present disclosureinclude promoters obtained from the genes for Aspergillus oryzae TAKAamylase, Rhizomucor miehei aspartic proteinase, Aspergillus nigerneutral alpha-amylase, Aspergillus niger acid stable alpha-amylase,Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucormiehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzaetriose phosphate isomerase, Aspergillus nidulans acetamidase, andFusarium oxysporum trypsin-like protease (see e.g., WO 96/00787, whichis hereby incorporated by reference herein), as well as the NA2-tpipromoter (a hybrid of the promoters from the genes for Aspergillus nigerneutral alpha-amylase and Aspergillus oryzae triose phosphateisomerase), and mutant, truncated, and hybrid promoters thereof.

In a yeast host, useful promoters can be from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP), andSaccharomyces cerevisiae 3-phosphoglycerate kinase. Other usefulpromoters for yeast host cells are described by Romanos et al., 1992,Yeast 8:423-488.

The control sequence may also be a suitable transcription terminatorsequence, a sequence recognized by a host cell to terminatetranscription. The terminator sequence is operably linked to the 3′terminus of the nucleic acid sequence encoding the polypeptide. Anyterminator which is functional in the host cell of choice may be used inthe present invention.

For example, exemplary transcription terminators for filamentous fungalhost cells can be obtained from the genes for Aspergillus oryzae TAKAamylase, Aspergillus niger glucoamylase, Aspergillus nidulansanthranilate synthase, Aspergillus niger alpha-glucosidase, and Fusariumoxysporum trypsin-like protease.

Exemplary terminators for yeast host cells can be obtained from thegenes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be a suitable leader sequence, anon-translated region of an mRNA that is important for translation bythe host cell. The leader sequence is operably linked to the 5′ terminusof the nucleic acid sequence encoding the polypeptide. Any leadersequence that is functional in the host cell of choice may be used.Exemplary leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase. Suitable leaders for yeast host cells areobtained from the genes for Saccharomyces cerevisiae enolase (ENO-1),Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomycescerevisiae alpha-factor, and Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′ terminus of the nucleic acid sequence andwhich, when transcribed, is recognized by the host cell as a signal toadd polyadenosine residues to transcribed mRNA. Any polyadenylationsequence which is functional in the host cell of choice may be used inthe present invention. Exemplary polyadenylation sequences forfilamentous fungal host cells can be from the genes for Aspergillusoryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillusnidulans anthranilate synthase, Fusarium oxysporum trypsin-likeprotease, and Aspergillus niger alpha-glucosidase. Usefulpolyadenylation sequences for yeast host cells are described by Guo andSherman, 1995, Mol Cell Bio 15:5983-5990.

The control sequence may also be a signal peptide coding region thatcodes for an amino acid sequence linked to the amino terminus of apolypeptide and directs the encoded polypeptide into the cell'ssecretory pathway. The 5′ end of the coding sequence of the nucleic acidsequence may inherently contain a signal peptide coding region naturallylinked in translation reading frame with the segment of the codingregion that encodes the secreted polypeptide. Alternatively, the 5′ endof the coding sequence may contain a signal peptide coding region thatis foreign to the coding sequence. The foreign signal peptide codingregion may be required where the coding sequence does not naturallycontain a signal peptide coding region.

Effective signal peptide coding regions for bacterial host cells are thesignal peptide coding regions obtained from the genes for Bacillus NC1B11837 maltogenic amylase, Bacillus stearothermophilus alpha-amylase,Bacillus licheniformis subtilisin, Bacillus licheniformisbeta-lactamase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiol Rev 57: 109-137.

Effective signal peptide coding regions for filamentous fungal hostcells can be the signal peptide coding regions obtained from the genesfor Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase,Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase,Humicola insolens cellulase, and Humicola lanuginosa lipase.

Useful signal peptides for yeast host cells can be from the genes forSaccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding regions are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding region that codesfor an amino acid sequence positioned at the amino terminus of apolypeptide. The resultant polypeptide is known as a proenzyme orpropolypeptide (or a zymogen in some cases). A propolypeptide isgenerally inactive and can be converted to a mature active polypeptideby catalytic or autocatalytic cleavage of the propeptide from thepropolypeptide. The propeptide coding region may be obtained from thegenes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilisneutral protease (nprT), Saccharomyces cerevisiae alpha-factor,Rhizomucor miehei aspartic proteinase, and Myceliophthora thermophilalactase (see e.g., WO 95/33836, which is hereby incorporated byreference herein).

Where both signal peptide and propeptide regions are present at theamino terminus of a polypeptide, the propeptide region is positionednext to the amino terminus of a polypeptide and the signal peptideregion is positioned next to the amino terminus of the propeptideregion.

It may also be desirable to add regulatory sequences, which allow theregulation of the expression of the polypeptide relative to the growthof the host cell. Examples of regulatory systems are those which causethe expression of the gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. In prokaryotic host cells, suitable regulatory sequencesinclude the lac, tac, and trp operator systems. In yeast host cells,suitable regulatory systems include, as examples, the ADH2 system orGAL1 system. In filamentous fungi, suitable regulatory sequences includethe TAKA alpha-amylase promoter, Aspergillus niger glucoamylasepromoter, and Aspergillus oryzae glucoamylase promoter.

Other examples of regulatory sequences are those which allow for geneamplification. In eukaryotic systems, these include the dihydrofolatereductase gene, which is amplified in the presence of methotrexate, andthe metallothionein genes, which are amplified with heavy metals. Inthese cases, the nucleic acid sequence encoding the transaminasepolypeptide of the present invention would be operably linked with theregulatory sequence.

Thus, in another embodiment, the present disclosure is also directed toa recombinant expression vector comprising a polynucleotide encoding anengineered transaminase polypeptide or a variant thereof, and one ormore expression regulating regions such as a promoter and a terminator,a replication origin, etc., depending on the type of hosts into whichthey are to be introduced. The various nucleic acid and controlsequences described above may be joined together to produce arecombinant expression vector which may include one or more convenientrestriction sites to allow for insertion or substitution of the nucleicacid sequence encoding the polypeptide at such sites. Alternatively, thenucleic acid sequence of the present disclosure may be expressed byinserting the nucleic acid sequence or a nucleic acid constructcomprising the sequence into an appropriate vector for expression. Increating the expression vector, the coding sequence is located in thevector so that the coding sequence is operably linked with theappropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus), which can be conveniently subjected to recombinant DNAprocedures and can bring about the expression of the polynucleotidesequence. The choice of the vector will typically depend on thecompatibility of the vector with the host cell into which the vector isto be introduced. The vectors may be linear or closed circular plasmids.

The expression vector may be an autonomously replicating vector, i.e., avector that exists as an extrachromosomal entity, the replication ofwhich is independent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one which, when introduced into thehost cell, is integrated into the genome and replicated together withthe chromosome(s) into which it has been integrated. Furthermore, asingle vector or plasmid or two or more vectors or plasmids whichtogether contain the total DNA to be introduced into the genome of thehost cell, or a transposon may be used.

The expression vector of the present invention preferably contains oneor more selectable markers, which permit easy selection of transformedcells. A selectable marker is a gene the product of which provides forbiocide or viral resistance, resistance to heavy metals, prototrophy toauxotrophs, and the like. Examples of bacterial selectable markers arethe dal genes from Bacillus subtilis or Bacillus licheniformis, ormarkers, which confer antibiotic resistance such as ampicillin,kanamycin, chloramphenicol, or tetracycline resistance. Suitable markersfor yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3.

Selectable markers for use in a filamentous fungal host cell include,but are not limited to, amdS (acetamidase), argB (ornithinecarbamoyltransferase), bar (phosphinothricin acetyltransferase), hph(hygromycin phosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),and trpC (anthranilate synthase), as well as equivalents thereof.Embodiments for use in an Aspergillus cell include the amdS and pyrGgenes of Aspergillus nidulans or Aspergillus oryzae and the bar gene ofStreptomyces hygroscopicus.

The expression vectors for expressing the transaminases can contain anelement(s) that permits integration of the vector into the host cell'sgenome or autonomous replication of the vector in the cell independentof the genome. For integration into the host cell genome, the vector mayrely on the nucleic acid sequence encoding the polypeptide or any otherelement of the vector for integration of the vector into the genome byhomologous or nonhomologous recombination.

Alternatively, the expression vector may contain additional nucleic acidsequences for directing integration by homologous recombination into thegenome of the host cell. The additional nucleic acid sequences enablethe vector to be integrated into the host cell genome at a preciselocation(s) in the chromosome(s). To increase the likelihood ofintegration at a precise location, the integrational elements shouldpreferably contain a sufficient number of nucleic acids, such as 100 to10,000 base pairs, preferably 400 to 10,000 base pairs, and mostpreferably 800 to 10,000 base pairs, which are highly homologous withthe corresponding target sequence to enhance the probability ofhomologous recombination. The integrational elements may be any sequencethat is homologous with the target sequence in the genome of the hostcell. Furthermore, the integrational elements may be non-encoding orencoding nucleic acid sequences. On the other hand, the vector may beintegrated into the genome of the host cell by non-homologousrecombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. Examples of bacterial origins of replication are P15Aori or the origins of replication of plasmids pBR322, pUC19, pACYC177(which plasmid has the P15A ori), or pACYC184 permitting replication inE. coli, and pUB110, pE194, pTA1060, or pAMβ1 permitting replication inBacillus. Examples of origins of replication for use in a yeast hostcell are the 2 micron origin of replication, ARS1, ARS4, the combinationof ARS1 and CEN3, and the combination of ARS4 and CEN6. The origin ofreplication may be one having a mutation which makes it's functioningtemperature-sensitive in the host cell (see, e.g., Ehrlich, 1978, ProcNatl Acad Sci. USA 75:1433).

More than one copy of a nucleic acid sequence of the present inventionmay be inserted into the host cell to increase production of the geneproduct. An increase in the copy number of the nucleic acid sequence canbe obtained by integrating at least one additional copy of the sequenceinto the host cell genome or by including an amplifiable selectablemarker gene with the nucleic acid sequence where cells containingamplified copies of the selectable marker gene, and thereby additionalcopies of the nucleic acid sequence, can be selected for by cultivatingthe cells in the presence of the appropriate selectable agent.

Many of the expression vectors for use in the present invention arecommercially available. Suitable commercial expression vectors includep3xFLAGTMTM expression vectors from Sigma-Aldrich Chemicals, St. LouisMo., which includes a CMV promoter and hGH polyadenylation site forexpression in mammalian host cells and a pBR322 origin of replicationand ampicillin resistance markers for amplification in E. coli. Othersuitable expression vectors are pBluescriptII SK(−) and pBK-CMV, whichare commercially available from Stratagene, LaJolla Calif., and plasmidswhich are derived from pBR322 (Gibco BRL), pUC (Gibco BRL), pREP4, pCEP4(Invitrogen) or pPoly (Lathe et al., 1987, Gene 57:193-201).

In another aspect, the present disclosure provides a host cellcomprising a polynucleotide encoding an improved transaminasepolypeptide of the present disclosure, the polynucleotide beingoperatively linked to one or more control sequences for expression ofthe transaminase enzyme in the host cell. Host cells for use inexpressing the transaminase polypeptides encoded by the expressionvectors of the present invention are well known in the art and includebut are not limited to, bacterial cells, such as E. coli, Lactobacillus,Streptomyces and Salmonella typhimurium cells; fungal cells, such asyeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCCAccession No. 201178)); insect cells such as Drosophila S2 andSpodoptera Sf9 cells; animal cells such as CHO, COS, BHK, 293, and Bowesmelanoma cells; and plant cells. Appropriate culture mediums and growthconditions for the above-described host cells are well known in the art.

Polynucleotides for expression of the transaminase may be introducedinto cells by various methods known in the art. Techniques include amongothers, electroporation, biolistic particle bombardment, liposomemediated transfection, calcium chloride transfection, and protoplastfusion. Various methods for introducing polynucleotides into cells willbe apparent to the skilled artisan.

An exemplary host cell is Escherichia coli W3110. The expression vectorwas created by operatively linking a polynucleotide encoding an improvedtransaminase into the plasmid pCK110900 operatively linked to the lacpromoter under control of the lad repressor. The expression vector alsocontained the P15a origin of replication and the chloramphenicolresistance gene. Cells containing the subject polynucleotide inEscherichia coli W3110 were isolated by subjecting the cells tochloramphenicol selection.

The improved transaminases and polynucleotides encoding suchpolypeptides can be prepared using methods commonly used by thoseskilled in the art. As noted above, the naturally-occurring amino acidsequence and corresponding polynucleotide encoding the transaminaseenzyme of Arthrobacter sp KNK168, from which the parent sequence SEQ IDNO:2 was derived, represented herein as SEQ ID NO:2) is available inU.S. Pat. No. 7,169,592, which is hereby incorporated by referenceherein. In some embodiments, the parent polynucleotide sequence is codonoptimized to enhance expression of the transaminase in a specified hostcell. The polynucleotide sequence designated SEQ ID NO: 1 was the parentsequence utilized as the starting point for most experiments and libraryconstruction of engineered transaminases.

The engineered transaminases can be obtained by subjecting thepolynucleotide encoding the naturally occurring transaminase tomutagenesis and/or directed evolution methods. An exemplary directedevolution technique is mutagenesis and/or DNA shuffling as described inStemmer, 1994, Proc Natl Acad Sci USA 91:10747-10751; WO 95/22625; WO97/0078; WO 97/35966; WO 98/27230; WO 00/42651; WO 01/75767 and U.S.Pat. No. 6,537,746 (each of which is hereby incorporated by referenceherein).

Other directed evolution procedures that can be used include, amongothers, staggered extension process (StEP), in vitro recombination (Zhaoet al., 1998, Nat. Biotechnol. 16:258-261), mutagenic PCR (Caldwell etal., 1994, PCR Methods Appl. 3:S136-S140), and cassette mutagenesis(Black et al., 1996, Proc Natl Acad Sci USA 93:3525-3529). Mutagenesisand directed evolution techniques useful for the purposes herein arealso described in the following references: Ling, et al., 1997,“Approaches to DNA mutagenesis: an overview,” Anal. Biochem.254(2):157-78; Dale et al., 1996, “Oligonucleotide-directed randommutagenesis using the phosphorothioate method,” Methods Mol. Biol.57:369-74; Smith, 1985, “In vitro mutagenesis,” Ann. Rev. Genet.19:423-462; Botstein et al., 1985, “Strategies and applications of invitro mutagenesis,” Science 229:1193-1201; Carter, 1986, “Site-directedmutagenesis,” Biochem. J. 237:1-7; Kramer et al., 1984, “Point MismatchRepair,” Cell 38:879-887; Wells et al., 1985, “Cassette mutagenesis: anefficient method for generation of multiple mutations at defined sites,”Gene 34:315-323; Minshull et al., 1999, “Protein evolution by molecularbreeding,” Curr Opin Chem Biol 3:284-290; Christians et al., 1999,“Directed evolution of thymidine kinase for AZT phosphorylation usingDNA family shuffling,” Nature Biotech 17:259-264; Crameri et al., 1998,“DNA shuffling of a family of genes from diverse species acceleratesdirected evolution,” Nature 391:288-291; Crameri et al., 1997,“Molecular evolution of an arsenate detoxification pathway by DNAshuffling,” Nature Biotech 15:436-438; Zhang et al., 1997, “Directedevolution of an effective fructosidase from a galactosidase by DNAshuffling and screening,” Proc Natl Acad Sci USA 94:45-4-4509; Crameriet al., 1996, “Improved green fluorescent protein by molecular evolutionusing DNA shuffling,’ Nature Biotech 14:315-319; and Stemmer, 1994,“Rapid evolution of a protein in vitro by DNA shuffling,” Nature370:389-391. All publications are incorporated herein by reference.

In some embodiments, the clones obtained following mutagenesis treatmentare screened for transaminases having a desired improved enzymeproperty. Measuring transaminase enzyme activity from the expressionlibraries can be performed using the standard techniques, such asseparation of the product (e.g., by HPLC) and detection of the productby measuring UV absorbance of the separated substrate and productsand/or by detection using tandem mass spectroscopy (e.g., MS/MS).Exemplary assays are described in Example 4 below. The rate of increasein desired product per unit time indicates the relative (enzymatic)activity of the transaminase polypeptide in a fixed amount of the lysate(or a lyophilized powder made therefrom). Where the improved enzymeproperty desired is thermal stability, enzyme activity may be measuredafter subjecting the enzyme preparations to a defined temperature andmeasuring the amount of enzyme activity remaining after heat treatments.Clones containing a polynucleotide encoding the desired transaminasesare then isolated, sequenced to identify the nucleotide sequence changes(if any), and used to express the enzyme in a host cell.

Where the sequence of the engineered polypeptide is known, thepolynucleotides encoding the enzyme can be prepared by standardsolid-phase methods, according to known synthetic methods. In someembodiments, fragments of up to about 100 bases can be individuallysynthesized, then joined (e.g., by enzymatic or chemical litigationmethods, or polymerase mediated methods) to form any desired continuoussequence. For example, polynucleotides and oligonucleotides of theinvention can be prepared by chemical synthesis using, e.g., theclassical phosphoramidite method described by Beaucage et al., 1981, TetLett 22:1859-69, or the method described by Matthes et al., 1984, EMBOJ. 3:801-05, e.g., as it is typically practiced in automated syntheticmethods. According to the phosphoramidite method, oligonucleotides aresynthesized, e.g., in an automatic DNA synthesizer, purified, annealed,ligated and cloned in appropriate vectors. In addition, essentially anynucleic acid can be obtained from any of a variety of commercialsources, The Great American Gene Company, Ramona, Calif., ExpressGenInc. Chicago, Ill., Operon Technologies Inc., Alameda, Calif., and manyothers.

The engineered transaminase enzymes expressed in a host cell can berecovered from the cells and or the culture medium using any one or moreof the well known techniques for protein purification, including, amongothers, lysozyme treatment, sonication, filtration, salting-out,ultra-centrifugation, and chromatography. Suitable solutions for lysingand the high efficiency extraction of proteins from bacteria, such as E.coli, are commercially available under the trade name CelLytic BTM fromSigma-Aldrich of St. Louis Mo.

Chromatographic techniques for isolation of the transaminase polypeptideinclude, among others, reverse phase chromatography high performanceliquid chromatography, ion exchange chromatography, gel electrophoresis,and affinity chromatography. Conditions for purifying a particularenzyme will depend, in part, on factors such as net charge,hydrophobicity, hydrophilicity, molecular weight, molecular shape, etc.,and will be apparent to those having skill in the art. In someembodiments, the engineered transaminases can be expressed as fusionproteins with purification tags, such as His-tags having affinity formetals, or antibody tags for binding to antibodies, e.g., myc epitopetag.

In some embodiments, affinity techniques may be used to isolate theimproved transaminase enzymes. For affinity chromatography purification,any antibody which specifically binds the transaminase polypeptide maybe used. For the production of antibodies, various host animals,including but not limited to rabbits, mice, rats, etc., may be immunizedby injection with an engineered polypeptide. The polypeptide may beattached to a suitable carrier, such as BSA, by means of a side chainfunctional group or linkers attached to a side chain functional group.Various adjuvants may be used to increase the immunological response,depending on the host species, including but not limited to Freund's(complete and incomplete), mineral gels such as aluminum hydroxide,surface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanin,dinitrophenol, and potentially useful human adjuvants such as BCG(bacilli Calmette Guerin) and Corynebacterium parvum.

EXAMPLES

Various features and embodiments of the disclosure are illustrated inthe following representative examples, which are intended to beillustrative, and not limiting.

Example 1 Wild-Type Transaminase Gene Acquisition and Construction ofExpression Vectors

Transaminase (TA) encoding genes were designed for expression in E.coli. based on the reported amino acid sequence of the transaminase, anda codon optimization algorithm as described in Example 1 of USapplication publication 20080248539, which is hereby incorporated byreference herein. Genes were synthesized using oligonucleotides,generally composed of 42 nucleotides, and the gene cloned into theexpression vector pCK110700 (depicted as FIG. 1 in US applicationpublication 20050153417, which is hereby incorporated by referenceherein) or pCK110900 (depicted as FIG. 3 in US application publication20060195947, which is hereby incorporated by reference herein) under thecontrol of a lac promoter. This expression vector also contains the P15aorigin of replication and the chloramphenicol resistance gene. Resultingplasmids were transformed into E. coli. W3110 using standard methods.Codon optimized genes and the encoding polypeptides are listed in Table2, and their sequences provided as SEQ ID NOs:1 and 2.

Likewise, the genes encoding the engineered transaminases of the presentdisclosure listed in Table 2 (SEQ ID NOs: 3-168) were cloned into vectorpCK110700 or pCK110900 for expression in E. coli W3110.

Example 2 Production of Transaminase Powders—Shake Flask Procedure

A single microbial colony of E. coli containing a plasmid encoding atransaminase of interest was inoculated into 50 mL Luria Bertani brothcontaining 30 μg/mL chloramphenicol and 1% glucose. Cells were grownovernight (at least 16 hrs) in an incubator at 30° C. with shaking at250 rpm. The culture was diluted into 250 mL M9YE (1.0 g/L ammoniumchloride, 0.5 g/L of sodium chloride, 6.0 g/L of disodium monohydrogenphosphate, 3.0 g/L of potassium dihydrogen phosphate, 2.0 g/L ofTastone-154 yeast extract, 1 L/L de-ionized water) containing 30 μg/mLchloramphenicol and 100 μM pyridoxine, in a 1 liter flask to an opticaldensity at 600 nm (OD600) of 0.2 and allowed to grow at 30° C.Expression of the transaminase gene was induced by addition of isopropylβ D-thiogalactoside (IPTG) to a final concentration of 1 mM when theOD600 of the culture is 0.6 to 0.8 and incubation was then continuedovernight (at least 16 hrs). Cells were harvested by centrifugation(5000 rpm, 15 min, 4° C.) and the supernatant discarded. The cell pelletwas resuspended with an equal volume of cold (4° C.) 100 mMtriethanolamine (chloride) buffer, pH 7.5 containing 100 μM pyridoxal5′-phosphate (PLP), and harvested by centrifugation as above. The washedcells were resuspended in two volumes of the cold triethanolamine(chloride) buffer containing PLP and passed through a French Press twiceat 12,000 psi while maintained at 4° C. Cell debris was removed bycentrifugation (9000 rpm, 45 min, 4° C.). The clear lysate supernatantwas collected and stored at −20° C. Lyophilization of frozen clearlysate provides a dry powder of crude transaminase enzyme.Alternatively, the cell pellet (before or after washing) may be storedat 4° C. or 80° C.

Example 3 Production of Transaminase—Fermentation Procedure

A single microbial colony of E. coli. containing a plasmid with thetransaminase gene of interest was inoculated into 2 mL M9YE broth (1.0g/L ammonium chloride, 0.5 g/L of sodium chloride, 6.0 g/L of disodiummonohydrogen phosphate, 3.0 g/L of potassium dihydrogen phosphate, 2.0g/L of Tastone-154 yeast extract, 1 L/L de-ionized water) containing 30μg/ml chloramphenicol and 1% glucose. Cells were grown overnight (atleast 12 hrs) in an incubator at 37° C. with shaking at 250 rpm. Afterovernight growth, 0.5 mL of this culture was diluted into 250 ml M9YEBroth, containing 30 μg/ml chloramphenicol and 1% glucose in 1 literflask and allowed to grow at 37° C. with shaking at 250 rpm. When theOD600 of the culture is 0.5 to 1.0, the cells were removed from theincubator and either used immediately, or stored at 4° C.

Bench-scale fermentations were carried out at 30° C. in an aerated,agitated 15 L fermentor using 6.0 L of growth medium (0.88 g/L ammoniumsulfate, 0.98 g/L of sodium citrate; 12.5 g/L of dipotassium hydrogenphosphate trihydrate, 6.25 g/L of potassium dihydrogen phosphate, 3.3g/L of Tastone-154 yeast extract, 0.083 g/L ferric ammonium citrate, and8.3 ml/L of a trace element solution containing 2 g/L of calciumchloride dihydrate, 2.2 g/L of zinc sulfate heptahydrate, 0.5 g/Lmanganese sulfate monohydrate, 1 g/L cuprous sulfate heptahydrate, 0.1g/L ammonium molybdate tetrahydrate and 0.02 g/L sodium tetraborate. Thevessel was sterilized at 121° C. and 15 PSI for 30 minutes, and 100 μMpyridoxine was added post sterilization. The fermentor was inoculatedwith a late exponential culture of E. coli W3110 containing a plasmidencoding the transaminase gene of interest (grown in a shake flask asdescribed above to a starting OD₆₀₀ of 0.5 to 1.0. The fermentor wasagitated at 250-1250 rpm and air was supplied to the fermentation vesselat 0.6-25 L/min to maintain a dissolved oxygen level of 50% saturationor greater. The pH of the culture was maintained at 7.0 by addition of20% v/v ammonium hydroxide. Growth of the culture was maintained byaddition of a feed solution containing 500 g/L Cerelose dextrose, 12 g/Lammonium chloride and 5.1 g/L magnesium sulfate heptahydrate. After theculture reached an OD₆₀₀ of 70+−10, expression of transaminase wasinduced by addition of isopropyl-β-D-thiogalactoside (IPTG) to a finalconcentration of 1 mM and fermentation is continued for another 18hours. The culture was then chilled to 4° C. and maintained at thattemperature until harvested. Cells were collected by centrifugation at5000 G for 40 minutes in a Sorval RC12BP centrifuge at 4° C. Harvestedcells were used directly in the following downstream recovery process orthey may be stored at 4° C. or frozen at −80° C. until such use.

The cell pellet was resuspended in 2 volumes of 100 mM triethanolamine(chloride) buffer, pH 7.5 containing 100 μM pyridoxal 5′-phosphate(PLP), at 4° C. to each volume of wet cell paste. The intracellulartransaminase was released from the cells by passing the suspensionthrough a homogenizer fitted with a two-stage homogenizing valveassembly using a pressure of 12000 psig. The cell homogenate was cooledto −20° C. immediately after disruption. A solution of 11% w/vpolyethyleneimine pH 7.2 was added to the lysate to a finalconcentration of 0.5% w/v. A solution of 1M Na₂SO₄ was added to thelysate to a final concentration of 100 mm. The lysate was then stirredfor 30 minutes. The resulting suspension was clarified by centrifugationat 5000 G in a Sorval RC12BP centrifuge at 4° C. for 30 minutes. Theclear supernatant was decanted and concentrated ten-fold using acellulose ultrafiltration membrane with a molecular weight cut off of 30kD. The final concentrate was dispensed into shallow containers, frozenat −20° C. and lyophilized to powder. The transaminase powder was storedat −80° C.

Example 4 High-Throughput Screening for Identification of Variants ofthe Arthrobacter Sp. KNK168 Transaminase Capable of StereoselectivelyConverting the Sitagliptin Ketoamide Substrate to Sitagliptin

Achiral HPLC method to determine conversion of the sitagliptin ketoamidesubstrate to sitagliptin: Enzymatic conversion of sitagliptin ketoamidesubstrate (prepared as described in U.S. Pat. No. 7,326,708) tositagliptin was determined using an Agilent 1200 HPLC equipped with anAgilent Eclipse XDB-C8 column (4.6×150 mm, 5 μm), using 45:55 10 mMNH₄Ac/MeCN as eluent at a flow rate of 1.5 ml/min and a columntemperature 40° C. Retention times: sitagliptin ketoamide substrate: 1.4min; sitagliptin: 1.7 min The sitagliptin ketoamide substrate andproduct in the eluant were determined as the peak area at 210 nm or 286nm, with a path length of 1 cm. Using these conditions, the limit ofdetection for sitagliptin was 5 μg/mL. Generally, an incident wavelengthof 210 nm was used for activity measurements for transaminases withactivity similar or equal to SEQ ID NO:4.

Chiral HPLC method to determine stereopurity of sitagliptin:Stereoisomeric purity of sitagliptin was determined using an Agilent1200 HPLC equipped with a Daicel Chiralpak AD-H column (4.6×150 mm, 5μm) using 60:40:0.1:0.1 EtOH/Heptane/diethylamine/water as the eluent ata flow rate of 0.8 ml/min and a column temperature of 35° C. Retentiontimes: sitagliptin ketoamide substrate: 6.3 min; (S)-enantiomer: 8.4min; sitagliptin: 10.8 min. The sitagliptin ketoamide substrate andproduct were determined as the peak area at 210 nm or 268 nm with a pathlength of 1 cm.

Liquid chromatography-mass spectroscopy (LC/MS) method for detectinglow-level conversion of the sitagliptin ketoamide substrate tositagliptin: Low-level enzymatic conversion of sitagliptin ketoamidesubstrate to sitagliptin was determined using an LC/MS/MS method. Fivemicroliters of sample was loaded into an Eclipse XDB-C8 HPLC column(4.6×150 mm) and eluted isocratically with a 40:60 mobile phase of 0.2%ammonium formate and methanol at 1.0 mL/min The retention time ofsitagliptin was 1.5 minutes at 35° C. Mass spectrometry was used fordetection on a Waters Quattro triple quadruple. Q1 was set to pass theM+H ion at 408.1 AMU and Q3 was set to pass the 235.1 daughter ion. Thecollision cell (Q2) had a collision energy of 17.0 and Argon gas flow of0.3 mL/min. Ionization was by APCI with a corona discharge of 5 μA,source temperature of 130° C. and probe temperature of 600° C.Desolvation gas flow was 100 L/minute and the cone gas was set to 50L/minute. Using these conditions the limit of detection for sitagliptinwas 71 pg/mL.

Example 5 High-Throughput Screening for Identification of Variants ofthe Arthrobacter Sp. KNK168 Transaminase Capable of StereoselectivelyConverting Sitagliptin Ketoamide Substrate to Sitagliptin

The gene encoding transaminase, constructed as described in Example 1,was mutagenized using methods described above and the population ofaltered DNA molecules was used to transform a suitable E. coli hoststrain. Antibiotic resistant transformants were selected and processedto identify those expressing a transaminase with an improved ability totransaminate the sitagliptin ketoamide substrate stereoselectively tositagliptin in the presence of a suitable amino group donor (i.e.,isopropylamine). Cell selection, growth, induced expression oftransaminase variant enzymes and collection of cell pellets were asdescribed below.

Recombinant E. coli colonies carrying a gene encoding transaminase werepicked using a Q-Bot® robotic colony picker (Genetix USA, Inc., Boston,Mass.) into 96-well shallow well microtiter plates containing in eachwell 180 μL LB Broth, 1% glucose and 30 μg/mL chloramphenicol (CAM).Cells were grown overnight at 30° C. with shaking at 200 rpm. A 10 μLaliquot of this culture was then transferred into 96-deep well platescontaining 390 μL M9YE broth, 100 μM pyridoxine and 30 μg/mL CAM. Afterincubation of the deep-well plates at 30° C. with shaking at 250 rpm for2-3 hrs, recombinant gene expression within the cultured cells wasinduced by addition of IPTG to a final concentration of 1 mM. The plateswere then incubated at 30° C. with shaking at 250 rpm for 18 hrs.

Cells were pelleted by centrifugation (4000 RPM, 10 min, 4° C.),resuspended in 200 μL lysis buffer and lysed by shaking at roomtemperature for 2 hours. The lysis buffer contained 100 mMtriethanolamine (chloride) buffer, pH 7.5 or 8.5, 1 mg/mL lysozyme, 500μg/mL polymixin B sulfate (PMBS) and 250 μM PLP. After sealing theplates with aluminum/polypropylene laminate heat seal tape (Velocity 11,Menlo Park, Calif., Cat #06643-001), they were shaken vigorously for 2hours at room temperature. Cell debris was pelleted by centrifugation(4000 RPM, 10 min, 4° C.) and the clear supernatant assayed directly orstored at 4° C. until use.

For screening in methanol or DMSO at pH 7.5, with early-stage engineeredtransaminases (i.e., early-stage “evolvants”), a 10 μL aliquot of asolution of sitagliptin ketoamide substrate (40 mg/mL) in methanol orDMSO was added to each well of a Costar® deep well plate, followed byaddition of 90 μL of 1.1 M isopropylamine hydrochloride using a BiomekNXp robotic instrument (Beckman Coulter, Fullerton, Calif.). This wasthen followed by addition of 100 μL of the recovered lysate supernatant,also performed using the Biomek NXp, to provide a reaction comprising of2 mg/ml sitagliptin ketoamide substrate, 500 mM isopropyl aminehydrochloride, 50 mM triethanolamine pH 7.5, and 5% methanol or DMSO(v/v). The plates were heat-sealed with aluminum/polypropylene laminateheat seal tape (Velocity 11, Menlo Park, Calif., Cat #06643-001) at 175°C. for 2.5 seconds and then shaken overnight (at least 16 hours) at 30°C. Reactions were quenched by the addition of 1 ml acetonitrile using aPhoenix Liquid Handling System (Art Robbins Instruments, Sunnyvale,Calif.). Plates were resealed, shaken for 5 min, and then centrifuged at4000 rpm for 10 min. A 200 μL aliquot of the cleared reaction mixturewas transferred to a new shallow well polypropylene plate (Costar#3365), sealed and analyzed as described in Example 4.

For screening in DMSO at pH 8.5 with late-stage engineered transaminases(i.e., late-stage “evolvants”), a 50 μL aliquot of a solution ofsitagliptin ketoamide substrate (400 mg/mL) in dimethyl sulfoxide (DMSO)was added to each well of a Costar® deep well plate, followed byaddition of 50 μL of 4 M isopropylamine hydrochloride using a Biomek NXrobotic instrument (Beckman Coulter, Fullerton, Calif.). This was thenfollowed by addition of 100 μL of the recovered lysate supernatant, alsoperformed using the Biomek NX, to provide a reaction comprising of 100mg/ml sitagliptin ketoamide substrate, 1 M isopropyl aminehydrochloride, 50 mM triethanolamine pH 8.5, and 25% DMSO (v/v). Theplates were heat-sealed with aluminum/polypropylene laminate heat sealtape (Velocity 11, Menlo Park, Calif., Cat #06643-001) at 175° C. for2.5 seconds and then shaken overnight (at least 16 hours) at 45° C.Reactions were quenched by the addition of 1 ml acetonitrile using aPhoenix Liquid Handling System (Art Robbins Instruments, Sunnyvale,Calif.). Plates were resealed, shaken for 5 min, and then centrifuged at4000 rpm for 10 min. A 10 μL aliquot of the cleared reaction mixture wastransferred to a new shallow well polypropylene plate (Costar #3365)containing 190 μL acetonitrile, sealed and analyzed as described inExample 4.

The transaminase of SEQ ID NO:2 expressed as in Examples 1 and 2exhibited no detectable activity on the sitagliptin ketoamide substrateusing the detection methods of Example 4. Variants of the Arthrobactersp. KNK168 transaminase capable of converting sitagliptin ketoamidesubstrate to sitagliptin were identified using the approaches andprocedures disclosed above. Multiple iterations of these processes, inwhich one or more improved isolates from one round were used as thestarting material for subsequent rounds of mutagenesis and screening,were used to develop or “evolve” Arthrobacter sp. KNK168 transaminasevariants with an improved ability to reduce the sitagliptin ketoamidesubstrate stereoselectively to sitagliptin.

Example 6 Stereoselective Transamination in Methanol of the SitagliptinKetoamide Substrate by Engineered Transaminases in Table 2 Derived fromArthrobacter Sp. KNK168 Transaminase

Improved transaminases designated “+” in Table 2 derived fromArthrobacter sp. KNK168 transaminase were evaluated at preparative scalein DMSO as follows. A 500 μL solution of transaminase variant (20 mg/mL)in 100 mM triethanolamine-chloride buffer pH 7.5 with 250 μM pyridoxal5′-phosphate was added to 5 mL reaction vial equipped with a magneticstir bar. Subsequently, 450 μL of 1.1 M isopropylamine hydrochloride,followed by 50 μL of a solution of sitagliptin ketoamide substrate (40mg/mL) in DMSO was added to the transaminase solution. The reaction wasstirred at 22° C. and monitored by HPLC analysis of samples takenperiodically from the reaction mixture (see Example 4 for analyticalconditions). Table 2 provides the SEQ ID NO. corresponding totransaminase variants designated “+”, the number of amino acid residuedifferences from the wild-type transaminase, and activity of each towardsitagliptin ketoamide substrate relative to that of the enzyme havingthe amino acid sequence of SEQ ID NO: 4.

The improved transaminases designated “++”, “+++”, “++++”, and “+++++”in Table 2 were assayed with conditions adjusted as follows: “++”: 2 g/Lsitagliptin ketoamide substrate, 0.5 M isopropylamine, 22° C., pH 7.5,5% MeOH; “+++”: 5-10 g/L sitagliptin ketoamide substrate, 0.5-1 Misopropylamine, 22-30° C., pH 7.5, 5% MeOH; “++++”: 10-40 g/Lsitagliptin ketoamide substrate, 1 M isopropylamine, 30-45° C., pH 8.5,10% MeOH; “+++++”: 40-100 g/L sitagliptin ketoamide substrate, 1 Misopropylamine, 45° C., pH 8.5, 10% MeOH-25% DMSO; and “++++++”: 40-100g/L ketoamide substrate, 1 M isopropylamine, 45° C., pH 8.5, 50% DMSO,1000 μM PLP. Relative activities of the improved transaminasesdesignated “+++”, “++++”, “+++++”, “++++++” were determined relative tothe activities of SEQ ID NO: 22, SEQ ID NO: 48, SEQ ID NO: 58, and SEQID NO:104, respectively.

For many engineered transaminases, conversion of sitagliptin ketoamidesubstrate to sitagliptin can also be achieved using amino group donorssuch as D-alanine, 3-aminobutyric acid, or α-methylbenzylamine at asuitable concentration.

Example 7 Preparation of (S)-2,2,2-trifluoro-1-phenylethanamine from2,2,2-trifluoro-1-phenylethanone

Preparation of (S)-2,2,2-trifluoro-1-phenylethanamine is illustrated asfollows:

Process. 1.4 g of isopropyl amine hydrochloride was added to 14 mL of0.1M triethanolamine buffer at pH 8.5. After dissolving theisopropylamine hydrochloride, 20 mg of PLP and 100 mg of a transaminaseof SEQ ID NO:74 were added and dissolved with gentle agitation at 400rpm. The reactor was heated to 60° C. and the pH of the solution wasadjusted to pH 8.5 with 5N NaOH. About 400 mg of2,2,2-trifluoro-1-phenylethanone ketone substrate was dissolved in 6 mLof DMSO and added dropwise to the solution over 2 hours. The reactor wasthen stirred at 500 RPM, 60° C., and pH set at 8.5 for 24 h. After 24 h,the reaction had reached 99% conversion to the(S)-2,2,2-trifluoro-1-phenylethanamine product. During workup, thetemperature of the reaction was decreased to 45° C. and 2 N HCl wasadded dropwise to decrease the pH of the reaction to pH 2. The reactionwas allowed to stir for 1 hour and the precipitate was filtered througha glass fritted funnel equipped with cotton towel. The precipitate waswashed three times with 10 mL of 0.1 N HCl. The aqueous filtrates werecombined and the pH was increased to pH 11 with 5 N NaOH, followed byextraction with 2×100 mL of IPAC. IPAC layers were washed with 25 mLbrine, dried with MgSO₄, filtered, and concentrated to an oil of the(S)-2,2,2-trifluoro-1-phenylethanamine product.

Example 8 Preparation of (R)-2-(2-fluorophenyl)pyrrolidine from4-chloro-1-(2-fluorophenyl)butan-1-one

Preparation of (R)-2-(2-fluorophenyl)pyrrolidine is illustrated asfollows:

Process. To a HPLC vial added charged 10 μL of ketone and 200 μL DMSO.To a 50 mL Falcon tube was added 3.75 g Isopropylamine-HCl and 30 mL of0.1M TEA buffer. About 37.5 mg PLP is added and the reaction mixturevortexed to mix. To 15 mL Falcon tubes was added 25 mg of a transaminaseof SEQ ID NO:80. A 5 mL solution of PLP/buffer was added to the enzymecontaining tube and vortexed to dissolve the enzyme. 1.0 mL of theenzyme solution was added to the LC vial containing the4-chloro-1-(2-fluorophenyl)butan-1-one ketone substrate and DMSO and thevial placed at 45° C. and mixed at 1000 rpm on a thermomixer. Afterseveral days, the LCMS analysis showed a 53 LCAP conversion to productwith an M+1 mass of 166. Coinjection with an authentic standard of thedesired (R)-2-(2-fluorophenyl)pyrrolidine product confirmed identity ofthis peak. The reaction mixture was extracted with 1.0 mL EtOAC. Thesample was concentrated and then diluted with methanol. Assay by SFCusing ChiralPak AD-H column as stationary phase showed(R)-2-(2-fluorophenyl)pyrrolidine in an enantiomeric excess of 95%.

Example 9 Preparation of (R)-ethyl 3-amino-3-(pyridin-2-yl)propanoatefrom ethyl 3-oxo-3-(pyridin-2-yl)propanoate

Preparation of (R)-ethyl 3-amino-3-(pyridin-2-yl)propanoate isillustrated as follows:

Methods/Materials: The reaction was carried out in a 3L round bottomflask with pH monitor, overhead stirring, heating mantle, andthermocouple. About 100 g of the ethyl 3-oxo-3-(pyridin-2-yl)propanoateketoester substrate was dissolved in 800 mL DMSO which resulted in asolution that is green. About 4 g of vitamin-B6 (“PLP”) was prepared in1.2 L of 0.5M triethanolamine buffer, pH 8.4 with 100 g/Lisopropylamine-HCl. After addition, the pH was 8.3. The pH was adjustedpH to 8.8 by addition of 3 mL 20 wt % KOH. The transaminase polypeptideof SEQ ID NO:86 (2 g) was prepared in buffer and mixed until fullydissolved. The pH of the solution is 8.77 and the solution kept at 21.7°C. The ketoester substrate prepared in DMSO stock was added directlyinto batch in one portion.

Reaction: The reaction is exothermic and heats the batch temp to 38.1°C. while the pH of solution is 8.45 and appears as a greenish slurry.The solution is heated to 50° C. At a temperature of 47° C., the pH ofsolution is 8.31, and the pH was then adjusted to 8.6 by adding 2 mL 20wt % KOH. After 2 hr from substrate charge, the pH was 8.07, and the pHwas adjusted to 9.02 by adding 4 mL of 4M isopropylamine solution. 6 hrafter the substrate charge, the pH was 8.05, and the pH was adjusted to8.9 by adding 4 mL of 4 M isopropylamine solution. The reaction wasallowed to incubate overnight. After 15 hr from substrate charge, the pHwas 7.4, and 47.6 mL of water added and the stirring increased (volumewas reduced ˜25%). About 8 mL of 4M isopropylamine was added to adjustthe pH to 8.85. After 17.25 hr, the pH was 8.5, and the reaction wasallowed to proceed without further adjustment of pH. At 18.33 hr, the pHwas 8.27 and the reaction was complete as determined by assay onRP-HPLC.

Reaction Workup: 2 g Solka Floc® was added to the solution at roomtemperature. The pH of the solution increased to 9.2 upon cooling asthere was a temperature dependent pH shift with the buffer. The pH ofthe solution was adjusted to 1.8 with addition of 4.5 mL concentratedH₂SO₄ and aged 1 hr. The solution was then vacuum filtered through a 5μm filter cloth with a filtering flask and fitted filter (60 mL-40M).The filtration took 1.5 hr. The cake was washed with 50.6 g of diluteH₂SO₄ (pH 1.6) by physically mixing with wash solution. The filtrationtook about 20 min. The cake was washed again with 50 g of dilute acidicsolution followed by rapid filtration in <5 min. The first wash andfirst acidic aqueous solutions were combined and 67 mL heptane and 3.3mL toluene added in one portion. The solution was mixed thoroughly andthe layers separated in a separation funnel. The first acidic aqueous 1results in about 63% recovery of the theoretical yield of combined(R)-ethyl 3-amino-3-(pyridin-2-yl)propanoate amino acid/aminoesterproducts. Acidic wash 1 results in about 27% recovery while the secondacidic wash results in about 10% recovery.

About 99.4 g of 20 wt % KOH was added to the solution to adjust the pHto 13 and the solution was incubated at 50° C. After 20 min, anadditional 11.8 g of 20 wt % KOH was added to adjust the pH from 12.1 to13. After an additional 20 min, the pH stabilized at 12.8 and hydrolysiswas complete as determined by HPLC. Solids were observed precipitatingout of solution. The basic solution was filtered on a fritted funnel(solids were dissolved in water and appeared to be inorganic as onlysmall amount of product was observed, the solids did not go into MeCN).The basic solution was concentrated on roto-evaporator to yield a crudepotassium-salt solution of (R)-ethyl 3-amino-3-(pyridin-2-yl)propanoatein 75% yield.

All publications, patents, patent applications and other documents citedin this application are hereby incorporated by reference in theirentireties for all purposes to the same extent as if each individualpublication, patent, patent application or other document wereindividually indicated to be incorporated by reference for all purposes.

While various specific embodiments have been illustrated and described,it will be appreciated that various changes can be made withoutdeparting from the spirit and scope of the invention(s).

What is claimed is:
 1. A process for preparing the compound of formula(III):

having the indicated stereochemical configuration at the stereogeniccenter marked with an * in an enantiomeric excess over the oppositeenantiomer, wherein, R¹⁰ is Cl, Br, F, CH₃, CF₃, CN, SO₂, —OCH₃, or NO₂,the process comprising: (a) contacting a ketone substrate of formula:

wherein R^(a) is halogen, OH, —C(O)R⁴, —OC(O)R⁵, or NR⁶R⁷, wherein R⁴,R⁵, R⁶, and R⁷ is H or C₁-C₄ alkyl, and R¹⁰ is as defined above, with atransaminase polypeptide in presence of an amino donor under reactionconditions suitable for converting the ketone substrate to an amineproduct of formula:

wherein the transaminase polypeptide has at least 80% sequence identityto SEQ ID NO:74 and is capable of converting the ketone substrate to theamine product at a rate that is improved as compared to SEQ ID NO:2; and(b) cyclizing the amine product under suitable conditions to form thecompound of formula (III).
 2. The process of claim 1, wherein the ketonesubstrate is 4-chloro-1-(2-fluorophenyl)butan-1-one:

and the amine product is (S)-4-chloro-1-(2-fluorophenyl)butan-1-amine:

thereby forming (R)-2-(2-fluorophenyl)pyrrolidine:

in enantiomeric excess.
 3. The process of claim 1, wherein the reactioncondition comprises a temperature of 20° C. to 65° C.
 4. The process ofclaim 3, wherein the reaction condition comprises a temperature of 40°C. to 65° C.
 5. The process of claim 1, wherein the amine product isproduced in at least 90% enantiomeric excess.
 6. The process of claim 1,wherein the amine product is produced in at least 99% enantiomericexcess.
 7. The process of claim 1, wherein the amino donor is selectedfrom isopropylamine, alanine, 3-aminobutyric acid, or methylbenzylamine.8. The process of claim 7, wherein the amino donor is isopropylamine. 9.The process of claim 1, further comprising the step of removing acarbonyl by-product of the reaction.
 10. The process of claim 9, whereinthe amino donor is an amino acid and the carbonyl by-product is a ketoacid.
 11. The process of claim 10, wherein the carbonyl by-product has avapor pressure higher than water, and removal of the carbonyl byproductis by sparging with a non-reactive gas or by applying a vacuum.
 12. Theprocess of claim 11, wherein the non-reactive gas is nitrogen gas. 13.The process of claim 11, wherein the amino group donor is isopropylamineand the carbonyl by-product is acetone.
 14. The process of claim 1,wherein the reaction condition is from a pH of about 7.0 to a pH ofabout 11.0.
 15. The process of claim 14, wherein the pH is maintained byadding isopropylamine.
 16. The process of claim 1, wherein the reactioncondition comprises a solvent of dimethylsulfoxide (DMSO).
 17. Theprocess of claim 16, wherein the DMSO is between about 10% to about 40%(v/v).
 18. The process of claim 1, wherein the substrate is present at 5to 25 g/L.
 19. The process of claim 1, wherein the transaminasecomprises an amino acid sequence having a residue difference as comparedto SEQ ID NO:2 at one or more residue positions selected from: X4; X5;X8; X18; X25; X26; X27; X28; X30; X41; X42; X48; X49; X50; X54; X55;X60; X61; X62; X65; X81; X94; X96; X102; X117; X120; X124; X126; X136;X137; X138; X146; X148; X150; X152; X155; X156; X160; X163; X164; X169;X174; X178; X195; X199; X204; X208; X209; X211; X215; X217; X225; X230;X252; X269; X273; X282, X292; X297; X306; X321; and X329.
 20. Theprocess of claim 19, wherein the residue difference with a transaminasepolypeptide of SEQ ID NO:2 occurs at one or more residue positionsselected from: X62, X69, X122, X136, X137, X195, X199, X208, X209, X223,X225, X282, and X284.
 21. The process of claim 19, wherein the type ofamino acid residue at the position of the residue difference is selectedfrom: X4 is an aromatic residue, X8 is a constrained residue; X26 is anaromatic or constrained residue; X48 is a polar, acidic, aliphatic ornon-polar residue; X60 is an aromatic residue; X61 is an aromaticresidue; X62 is an aromatic or polar residue; X65 is an aliphaticresidue; X69 is a cysteine (C) or non-polar, polar, or aliphatic residueX81 is a non-polar residue; X94 is an aliphatic residue; X96 is analiphatic residue; X102 is an aliphatic or basic residue; X122 is aconstrained, non-polar or aliphatic residue; X124 is a polar orconstrained residue; X136 is an aromatic residue; X137 is a polar oraliphatic residue; X150 is aromatic, constrained or polar residue; X152is cysteine (C), non-polar, aliphatic, or polar residue; X160 is analiphatic residue; X163 is an aliphatic or constrained residue; X169 isan aliphatic residue; X174 is an aliphatic residue; X178 is a polarresidue; X195 is an aromatic or polar residue; X199 is an aliphatic oraromatic residue; X208 is cysteine (C) or constrained, non-polar,aromatic, polar, or basic residue; X209 is an aliphatic residue; X211 isan aliphatic residue; X215 is a cysteine (C); X217 is a polar residue;X223 is a constrained residue; X225 is an aromatic residue; X230 is analiphatic residue; X252 is an aromatic or aliphatic residue; X269 is aconstrained residue; X273 is an aromatic residue; X282 is a polarresidue; X284 is a non-polar residue X292 is a polar residue; X297 is apolar residue; X306 is an aliphatic residue; X321 is a constrainedresidue, and X329 is a constrained or aromatic residue.
 22. The processof claim 19, wherein the amino acid residue at the position of theresidue difference is selected from: X4 is Y; X8 is P; X26 is H; X48 isQ, D, V, G, or A; X60 is F; X61 is Y; X62 is T, Y or F; X65 is A; X69 isG, C, T, A, or S; X81 is G; X94 is I or L; X96 is L; X102 is L or K;X122 is M, I, L, V, or H; X124 T, H or N; X136 is Y or F; X137 is T orI; X150 is F, H, or S; X152 is I, L, S or C; X160 is L; X163 is H or V;X169 is L; X174 is A; X178 is S; X195 is F or Q; X199 is W or I; X208 isH, C, G, K, N, Y, D or S; X209 is L; X211 is I; X215 is C; X217 is N;X223 is P; X225 is Y; X230 is V; X252 is F; X269 is P; X273 is Y; X282is S; X284 is G; X292 is T; X297 is S; X306 is L; X321 is P; and X329 isH.
 23. The process of claim 22, wherein the amino acid residue at theposition of the residue difference is selected from: X8 is P; X60 is F;X61 is Y; X62 is T, Y or F; X65 is A; X69 is G, C, T, A, or S; X81 is G;X94 is I or L; X96 is L; X122 is M, I, L, V, or H; X124 T, H or N; X136is Y or F; X169 is L; X178 is S; X199 is W or I; X209 is L; X215 is C;X217 is N; X223 is P; X269 is P; X273 is Y; X282 is S; X284 is G; X297is S; X321 is P and X329 is H.
 24. The process of claim 19, wherein thetransaminase polypeptide has an amino acid sequence that corresponds tothe sequence of SEQ ID NO: 58, 72, 74, 80, 86, 96, 98, 100, or 102.